Minimally immunogenic variants of sdr-grafted humanized antibody cc49 and their use

ABSTRACT

Humanized anti-TAG-72 CC49 monoclonal antibodies are disclosed herein. The antibodies include a light chain Complementarity Determining Region (L-CDR)1, a L-CDR2, and a L-CDR3; and a heavy chain Complementarity Determining Region (H-CDR)1, a H-CDR2, and a H-CDR3 from humanized antibody HuCC49V10. The L-CDR1, L-CDR2, L-CDR3 are within a HuCC49V10 light chain framework region that includes the corresponding amino acid from LEN at position 5, 19, 21, and 106 in the light chain. The H-CDR1, H-CDR2, and H-CDR3 are within a heavy chain HuCC49V10 framework comprising a human 21/28′ CL residue at positions 20, 38, 48, 66, 67, 69, and 80 in the heavy chain. These humanized CC49 antibodies retain binding affinity for TAG-72 and have reduced immunogenicity, as compared to a parental HuCC49V10 antibody. Methods are disclosed herein for using these antibodies in the treatment or diagnosis of a tumor, such as a carcinoma, expressing TAG-72.

PRIORITY CLAIM

This application claims the benefit of U.S. Provisional Application No.60/498,903, filed Aug. 29, 2003, which is incorporated herein byreference.

FIELD

The present disclosure relates to humanized monoclonal antibodies thatbind a tumor antigen. More specifically, the present disclosure relatesto humanized monoclonal antibodies with non-conservative amino acidsubstitutions that have a high binding affinity for tumor-associatedglycoprotein (TAG)-72 and minimal immunogenicity.

BACKGROUND

The use of murine monoclonal antibodies in medicine has significantpotential especially in the diagnosis and treatment of various diseases,including cancer. The advantage of using monoclonal antibodies residesis their specificity for a single antigen. A monoclonal antibody raisedagainst a specific tumor cell surface antigen can be coupled totherapeutic agents, such as radioisotopes and chemotherapeutic drugs,and these immunoconjugates can be used clinically to specificallytarget, for example, a tumor cell of interest.

A major limitation in the clinical use of monoclonal antibodies is thedevelopment of a human anti-murine antibody (HAMA) response in thepatients receiving the treatments. The HAMA response can involveallergic reactions and an increased rate of clearance of theadministered antibody from the serum. Various types of modifiedmonoclonal antibodies have been developed to minimize the HAMA responsewhile trying to maintain the antigen binding affinity of the parentmonoclonal antibody. One type of modified monoclonal antibody is ahuman-mouse chimera in which a murine antigen-binding variable region iscoupled to a human constant domain (Morrison and Schlom, ImportantAdvances in Oncology, Rosenberg, S. A. (Ed.), 1989). A second type ofmodified monoclonal antibody is the complementarity determining region(CDR)-grafted, or humanized, monoclonal antibody (Winter and Harris,Immunol. Today 14:243-246, 1993). A more recent method in thehumanization procedure is based on grafting, onto the variable (VL) andvariable heavy (VH) frameworks of human monoclonal antibodies (mAb), ofthe specificity determining residues (SDRs), the CDR residues that arecrucial for the complementarity of the antigen (Ag):Ab surfaces(Kashmiri et al., Crit. Rev. Oncol. Hematol. 38: 3-16, 2001). Ingenerating humanized Abs, whether by grafting CDRs or SDRs, a few murineframework residues considered crucial for the maintenance of the Abcombining sites are also transplanted onto the human frameworks (forexample, see Abola et al., Methods Enzymol 277, 556-71, 1997).

Murine CC49 (mCC49) is an antibody that specifically recognizes atumor-associated glycoprotein (TAG)-72 expressed on a majority of humancarcinomas (Muraro et al., Cancer Res. 48:4588-4596, 1988). Theseantibodies have been shown to efficiently target colorectal (Divgi etal., J Nucl Med 36:586-592, 1995; Divgi et al., Clin Cancer Res1:1503-1510, 1995; Liu et al., Cancer Biother Radiopharm 12:79-87, 1997;Meredith et al., Clin Cancer Res 2:1811-1818, 1996; Rucker et al., JImmunother 22:80-84, 1999; Tempero et al., J Clin Oncol 15:1518-1528,1997), breast (Macey et al., Clin Cancer Res 3:1547-1555, 1997; Murrayet al., Cancer Res 55:5925s-5928s, 1995), ovarian (Alvarez et al.,Gynecol Oncol 65:94-101, 1997; Meredith et al., Cancer BiotherRadiopharm 16:305-315, 2001;

Meredith et al., J Nucl Med 37:1491-1496, 1996), and prostate (Liu etal., Cancer Biother Radiopharm 12-7987, 1997; Meredith et al., ClinCancer Res 5:3254s-3258s, 1999; Slovin et al., Clin Cancer Res4:643-651, 1998) carcinomas in several phase I/II clinical trials. As atherapeutic reagent, humanized CC49 (¹⁷⁷Lu-mCC49) has been found toevoke objective responses in ovarian cancer patients (Alvarez et aL,Gynecol Oncol 65:94-101, 1997; Meredith et al., Cancer BiotherRadiopharm 16:305-315, 2001; Meredith et al., J Nucl Med 37:1491-1496,1996), while objective responses have also been reported in metastaticbreast and prostate cancer patients administered with one or two dosesof ¹³¹I-mCC49 (Macey et al., Clin Cancer Res 30 3:1547-1555, 1997;Meredith et al., Clin Cancer Res 5:3254s-3258s, 1999). mCC49 has alsobeen used in radioimmunoguided surgery, which is more sensitive indetecting metastases than the traditional clinical and histologicalexaminations (Cote et al., Cancer 77:613-620, 1996; LaValle et al.,Surgery 122:867-871, 1997; McIntosh et al., Cancer Biother Radiopharm12:287-294, 1997), thus resulting in better disease staging (Haddad etal., Eur J Surg Oncol 27:298-301, 2001; Schneebaum et al., RecentResults Cancer Res 157:281-292, 2000).

Unfortunately, the clinical utility of the mCC49 monoclonal antibody hasbeen limited because of its murine origin. Thus, there clearly exists aneed to develop a humanized CC49 antibody with both high antigen bindingaffinity and low immunogenicity for use in human subjects.

SUMMARY

Humanized anti-TAG-72 CC49 monoclonal antibodies are disclosed herein.The antibodies are variants of a humanized CC49 antibody that arespecifically designed to minimize immunogenicity in human subjects. Theantibodies are also designed to retain or improve the bindingspecificity for TAG-72. In one example, the antibodies are produced bygenetic manipulation of the framework region of HuCC49V10.

In one embodiment, the antibodies include a light chain ComplementarityDetermining Region (L-CDR)1, an L-CDR2, and an L-CDR3; and a heavy chainComplementarity Determining Region (H-CDR)1, an H-CDR2, and an H-CDR3from humanized antibody HuCC49V10. The L-CDR1, L-CDR2, L-CDR3 are withina HuCC49V10 light chain framework region that includes the correspondingamino acid from LEN at position 5, 19, 21, and 106 in the light chain.The H-CDR1, H-CDR2, and H-CDR3 are within a heavy chain HuCC49V10framework comprising a human 21/28′ CL residue at positions 20, 38, 48,66, 67, 69, and 80 in the heavy chain. These humanized CC49 antibodiesretain binding affinity for TAG-72 and have reduced immunogenicity, ascompared to a parental Hu-CC49 V10 antibody.

In another embodiment, the antibodies include an L-CDR1 from HuCC49V14,and an L-CDR2, an L-CDR3, an H-CDR-1, an H-CDR2, and an H-CDR3 fromhumanized antibody HuCC49V10. The L-CDR1, L-CDR2, L-CDR3 are within aHuCC49V10 light chain framework region that includes the correspondingamino acid from LEN at position 5, 19, 21, and 106 in the light chain.The H-CDR1, H-CDR2, and H-CDR3 are within a heavy chain HuCC49V10framework comprising a human 21/28′ CL residue at positions 20, 38, 48,66, 67, 69, and 80 in the heavy chain. These humanized CC49 antibodiesretain binding affinity for TAG-72 and have reduced immunogenicity, ascompared to a parental HuCC49V10 antibody.

In a further embodiment, the antibodies include an L-CDR1 and an L-CDR3from HuCC49V15, an L-CDR2, an HCDR1, an H-CDR2, and an H-CDR3 fromhumanized antibody HuCC49V10. The L-CDR1, L-CDR2, L-CDR3 are within aHuCC49V10 light chain framework region that includes the correspondingamino acid from LEN at position 5, 19, 21, and 106 in the light chain.The H-CDR1, H-CDR2, and H-CDR3 are within a heavy chain HuCC49V10framework comprising a human 21/28′ CL residue at positions 20, 38, 48,66, 67, 69, and 80 in the heavy chain. These humanized CC49 antibodiesretain binding affinity for TAG-72 and have reduced immunogenicity, ascompared to a parental HuCC49V10 antibody.

Methods are disclosed herein for using these antibodies for treating atumor expressing TAG-72. Methods are also disclosed herein for usingthese antibodies to detect tumor cells expressing TAG-72.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-B are schematic diagrams of the generation of the expressionconstructs derived from the genes encoding the light (L) and heavy (H)chains of the framework variants of HuCC49V10. The pDCM-dhfr vector wasused to generate the expression construct of the L chain (pDCMdhfrV10 Lchain), as shown in FIG. 1A. Expression constructs of the frameworkvariants containing mutations in the L chain were generated by swappingthe appropriate PCR-amplified VL region sequences with the VL sequenceof HuCC49V10 in this DNA construct through the HindIII/SacI site, priorto insertion of the appropriate H chain sequence into the vector.Generation of the dual expression construct of the L and H chains ofHuCC49V10 (pDCMdhfrV10 L and H chains) is shown in FIG. 1B. To generateconstructs containing the genes of the H chain framework variants, thesuitable PCR-amplified VH sequences were exchanged with the HuCC49V10 VHsequence through the unique EcoRIlApaI site in the pDCMdhfr constructcontaining the L and H chains of HuCC49V10. CMV, human cytomegaloviruspromoter; BGH pA, bovine growth hormone polyadenylation signal sequence;Amp, ampicillinr; dhfr, dihydrofolate reductase gene; Neo, neomycinrgene; CL and CH, constant regions of human κ and γ1 chains.

FIG. 2 shows the amino acid sequences of the framework variants ofHuCC49V10. The complete amino acid sequence of all the frameworks of thehumanized Ab HuCC49V10 is given in the top row of each of the light (SEQID NOs: 1-4) and heavy chain (SEQ ID NOs: 5-8) panels. Letters in boldindicate the murine framework residues that were retained in theHuCC49V10. Dashes indicate residues that are identical among V10, V35,and V37, or V10, V40, and V41.

FIG. 3 is a digital image of an analysis of the purified recombinant Absusing the Agilent Bioanalyzer system under reducing conditions. Lane L,molecular weight (m.w.) markers; Lane 1, V10; Lane 2, V35; Lane 3, V37;Lane 4, V40; Lane 5, V41; Lane 6, V47; Lane 7, V48; Lane 8, V58; Lane 9,V59. Sizes of the m.w. markers are given in the column at the left.Three peaks are present in all lanes corresponding to the lower maker (6kDa), system peak (9 kDa) and upper marker (210 kDa).

FIG. 4 is a set of binding sensorgrams of V10 and V10-derived frameworkvariants to BSM immobilized onto the surface of a CM5 sensor chip.Binding was measured using a Biacore X system, as described in Materialsand Methods, at Ab concentrations of 10, 30, 90, 250, 500 and 1250 nM.

FIG. 5 is a graph of a competition RIA of V10-derived framework Abs.Increasing concentrations of Abs mCC49 (●), HuCC49 (▪), V10 (▴), V47(Δ), V59 (□), and HuIgG (◯) were used to compete for the binding of¹²⁵I-labeled mCC49 to 10 ng of BSM coated in each well.

FIGS. 6A-C are plots of flow cytometric analysis of the binding of V10and its variants to cells expressing cell surface TAG-72. Bindingprofiles of 0.5 μg of V10 (FIG. 6A), V47 (FIG. 6B) and V59 (FIG. 6C) toJurkat cells expressing TAG-72 on its cell surface. Binding of anirrelevant mAb, human IgG (dashed line) is shown in each panel andrepresents less than 1% of the cell population.

FIGS. 7A-B are two plots showing sera reactivity, by SPR, of V10 and itsframework variants. Increasing concentrations of HuCC49 (♦), V10 (▪),V47 (Δ), and V59 (◯) mAbs were used to compete with the anti-V regionAbs to CC49 present in the sera of patients EA (FIG. 7A) and DS (FIG.7B) for binding to HuCC49 immobilized on a sensor chip. Percent bindingof the sera to immobilized HuCC49 was calculated from the sensorgramsand plotted as a function of the concentration of the competitor.

FIG. 8 is a digital image showing a comparison of the CDR sequences ofmurine MAb CC49 and humanized MAb HuCC49 with the corresponding CDRsequences of human MAbs LEN and 21/28′ CL. Amino acid residues arenumbered using the convention of Kabat et al. (Sequences of Proteins ofImmnunological Interest, 5^(th) Edition, Department of Health and HumanServices, Public Health Service, National Institutes of Health, Bethesda(NIH Publication No. 91-3242), 1991) The underlined numbers indicate thespecificity determining residues (SDRs). CDR1, CDR2 and CDR3 within thelight chain of HuCC49 and CC49 correspond to SEQ ID NOs: 31-33,respectively (SEQ ID NOs: 1-3 of U.S. application Ser. No. 09/830,748,which is incorporated herein by reference). CDR1, CDR2 and CDR3 withinthe heavy chain of HuCC49 and CC49 correspond to SEQ ID NOs: 34-36,respectively (SEQ ID NOs: 4-6 of U.S. application Ser. No. 09/830,748,which is incorporated herein by reference). CDR1, CDR2 and CDR3 withinthe light chain of human antibody LEN correspond to SEQ ID NOs: 15-17,respectively. CDR1, CDR2 and CDR3 within the heavy chain of humanantibody 21/28′ CL correspond to SEQ ID NOs: 18-20, respectively (SEQ IDNOs: 10-12 of U.S. application Ser. No. 09/830,748, which isincorporated herein by reference). In HuCC49V10, amino acid 60 is aserine, amino acid 61 is a glutamine, amino acid 62 is a lysine, aminoacid 64 is a glutamine in the heavy chain, and amino acid 97 is athreonine in the light chain.

FIG. 9A shows the amino acid sequence of the VL frameworks of human MAbLEN (SEQ ID NOs: 21-24) (see also U.S. application Ser. No. 09/830,748,which is incorporated herein by reference), the entire sequence of theVL region of humanized CC49 (HuCC49, SEQ ID NO: 25) including theframeworks and CDRs, and the entire sequence of the VL region of themurine CC49 (mCC49, SEQ ID NO: 81), including frameworks and CDRs. FIG.9B shows the amino acid sequences of VH frameworks of human mAb 21/28′CL (SEQ ID NOs: 26-29), the entire sequence of the VH region of HuCC49,including the frameworks and CDRs (SEQ ID NO: 30), and the entiresequence of the VH region of the murine CC49 (SEQ ID NOs: 82). Thesequence of the mCC49 CDRs are individually set forth as SEQ NOs: 31-36.Framework residues previously deemed to be important in maintaining thecombining site structure of CC49 on the basis of structure are marked byan asterisk. Those murine framework residues deemed important forbinding, and that are different from the residue at the correspondingposition in the human sequences, were retained (see also U.S.application Ser. No. 09/830,748, which is incorporated herein byreference).

SEQUENCE LISTING

The amino acid sequences listed in the accompanying sequence listing areshown using standard letter abbreviations for nucleotide bases, andthree letter code for amino acids, as defined in 37 C.F.R. 1.822. Onlyone strand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of FR1 of the light chain ofHuCC49V10.

SEQ ID NO: 2 is the amino acid sequence of FR2 of the light chain ofHuCC49V10.

SEQ ID NO: 3 is the amino acid sequence of FR3 of the light chain ofHuCC49V10.

SEQ ID NO: 4 is the amino acid sequence of FR4 of the light chain ofHuCC49V10.

SEQ ID NO: 5 is the amino acid sequence of FR1 of the heavy chain ofHuCC49V10.

SEQ ID NO: 6 is the amino acid sequence of FR2 of the heavy chain ofHuCC49V10.

SEQ ID NO: 7 is the amino acid sequence of FR3 of the heavy chain ofHuCC49V10.

SEQ ID NO: 8 is the amino acid sequence of FR4 of the heavy chain ofHuCC49V10.

SEQ ID NO: 9 is the amino acid sequence of the LCDR1 of HuCC49V10.

SEQ ID NO: 10 is the amino acid sequence of the LCDR2 of HuCC49V10.

SEQ ID NO: 11 is the amino acid sequence of the LCDR3 of HuCC49V10.

SEQ ID NO: 12 is the amino acid sequence of the HCDR1 of HuCC49V10.

SEQ ID NO: 13 is the amino acid sequence of the HCDR2 of HuCC49V10.

SEQ ID NO: 14 is the amino acid sequence of the HCDR3 of HuCC49V10.

SEQ ID NO: 15 is the amino acid sequence of the LCDR1 of LEN.

SEQ ID NO: 16 is the amino acid sequence of the LCDR2 of LEN.

SEQ ID NO: 17 is the amino acid sequence of the LCDR3 of LEN.

SEQ ID NO: 18 is the amino acid sequence of the HCDR1 of 21/28′ CL.

SEQ ID NO: 19 is the amino acid sequence of the HCDR2 of 21/28′ CL.

SEQ ID NO: 20 is the amino acid sequence of the HCDR3 of 21/28′ CL.

SEQ ID NO: 21 is the amino acid sequence of the light chain FR1 of LEN.

SEQ ID NO: 22 is the amino acid sequence of the light chain FR2 of LEN.

SEQ ID NO: 23 is the amino acid sequence of the light chain FR3 of LEN.

SEQ ID NO: 24 is the amino acid sequence of the light chain FR4 of LEN.

SEQ ID NO: 25 is the amino acid sequence of the variable light chain ofHuCC49.

SEQ ID NO: 26 is the amino acid sequence of the light chain FR1 of21/28′ CL.

SEQ ID NO: 27 is the amino acid sequence of the light chain FR2 of21/28′ CL.

SEQ ID NO: 28 is the amino acid sequence of the light chain FR3 of21/28′ CL.

SEQ ID NO: 29 is the amino acid sequence of the light chain FR4 of21/28′ CL.

SEQ ID NO: 30 is the amino acid sequence of the variable heavy chain ofHuCC49.

SEQ ID NO: 31 is the amino acid sequence of the LCDR1 of CC49 andHuCC49.

SEQ ID NO: 32 is the amino acid sequence of the LCDR2 of CC49 andHuCC49.

SEQ ID NO: 33 is the amino acid sequence of the LCDR3 of CC49 andHuCC49.

SEQ ID NO: 34 is the amino acid sequence of the HCDR1 of CC49 andHuCC49.

SEQ ID NO: 35 is the amino acid sequence of the HCDR2 of CC49 andHuCC49.

SEQ ID NO: 36 is the amino acid sequence of the HCDR3 of CC49 andHuCC49.

SEQ ID NO: 37 is the amino acid sequence of the FR1 of V35.

SEQ ID NO: 38 is the amino acid sequence of the FR2 of V35.

SEQ ID NO: 39 is the amino acid sequence of the FR3 of V35.

SEQ ID NO: 40 is the amino acid sequence of the FR4 of V35.

SEQ ID NO: 41 is the amino acid sequence of the FR1 of V37.

SEQ ID NO: 42 is the amino acid sequence of the FR2 of V37.

SEQ ID NO: 43 is the amino acid sequence of the FR3 of V37.

SEQ ID NO: 44 is the amino acid sequence of the FR4 of V37.

SEQ ID NO: 45 is the amino acid sequence of the FR1 of V40.

SEQ ID NO: 46 is the amino acid sequence of the FR2 of V40.

SEQ ID NO: 47 is the amino acid sequence of the FR3 of V40.

SEQ ID NO: 48 is the amino acid sequence of the FR4 of V40.

SEQ ID NO: 49 is the amino acid sequence of the FR1 of V41.

SEQ ID NO: 50 is the amino acid sequence of the FR2 of V41.

SEQ ID NO: 51 is the amino acid sequence of the FR3 of V41.

SEQ ID NO: 52 is the amino acid sequence of the FR4 of V41.

SEQ ID NOs: 53-72 are the nucleic acid sequence of primers.

SEQ ID NO: 73 is the amino acid sequence of the light chain FR1 of themurine CC49 (mCC49).

SEQ ID NO: 74 is the amino acid sequence of the light chain FR2 of themurine CC49 (mCC49).

SEQ ID NO: 75 is the amino acid sequence of the light chain FR3 of themurine CC49 (mCC49).

SEQ ID NO: 76 is the amino acid sequence of the light chain FR4 of themurine CC49 (mCC49).

SEQ ID NO: 77 is the amino acid sequence of the heavy chain FR1 of themurine CC49 (mCC49).

SEQ ID NO: 78 is the amino acid sequence of the heavy chain FR2 of themurine CC49 (mCC49).

SEQ ID NO: 79 is the amino acid sequence of the heavy chain FR3 of themurine CC49 (mCC49).

SEQ ID NO: 80 is the amino acid sequence of the heavy chain FR4 of themurine CC49 (mCC49).

SEQ ID NO: 81 is the amino acid sequence of the variable light chain ofmCC49.

SEQ ID NO: 82 is the amino acid sequence of the variable heavy chain ofmCC49.

DETAILED DESCRIPTION

I. Abbreviations

-   Ab antibody-   Ag antigen-   BSM bovine submaxillary mucin-   C constant-   CH constant heavy-   CHO Chinese hamster ovary-   CL constant light-   CDR complementarity determining region-   ELISA enzyme-linked immunosorbent assay-   Fab fragment antigen binding-   F(ab′)₂ Fab with additional amino acids, including cysteines    necessary for disulfidebonds-   FACS fluorescence activated cell sort-   FR framework region-   Fv fragment variable-   H heavy-   HAMA human antimurine antibody-   HuIgG human immunoglobulin G-   IC₅₀ half maximal inhibition of binding-   Ig immunoglobulin-   IL interleukin-   Ka relative affinity constant-   L light-   mCC49 murine CC49-   m.w. molecular weight-   PCR polymerase chain reaction-   PDB protein data bank-   RIA radioimmunoassay-   RU resonance unit-   scFv single chain Fv-   SDR specificity determining residue-   SPR surface plasmon resonance-   SSKI saturated solution of potassium iodide-   TAG-72 tumor associated glycoprotein-72-   TNF tumor necrosis factor-   V variable-   VH variable heavy-   VL variable light-   V10 HuCC49V10    II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: Immunoglobulin (Ig) molecules and immunologically activeportions of Ig molecules, for instance, molecules that contain anantigen binding site which specifically binds (immunoreacts with) anantigen.

A naturally occurring antibody (for example, IgG) includes fourpolypeptide chains, two heavy (H) chains and two light (L) chainsinterconnected by disulfide bonds. The two heavy chains are linked toeach other by disulfide bonds and each heavy chain is linked to a lightchain by a disulfide bond. There are two types of light chain, lambda(λ) and kappa (κ). There are five main heavy chain classes (or isotypes)which determine the functional activity of an antibody molecule: IgM,IgD, IgG, IgA and IgE.

Each chain contains distinct sequence domains. The light chain includestwo domains, a variable domain (VL) and a constant domain (CL). Theheavy chain includes four domains, a variable domain (VH) and threeconstant domains (CH1, CH2 and CH3, collectively referred to as CH). Thevariable regions of both light (VL) and heavy (VH) chains determinebinding recognition and specificity to the antigen. The constant regiondomains of the light (CL) and heavy (CH) chains confer importantbiological properties such as antibody chain association, secretion,transplacental mobility, complement binding, and binding to Fcreceptors. The specificity of the antibody resides in the structuralcomplementarity between the antibody combining site and the antigenicdeterminant. Antibody combining sites are made up of residues that areprimarily from the hypervariable or complementarity determining regions(CDRs). However, it is believed that residues from nonhypervariable orframework regions (FR) influence the overall domain structure and hencethe combining site.

It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a naturally occurring antibody. Thus, theseantigen-binding fragments are also intended to be designated by the term“antibody.” Examples of binding fragments encompassed within the termantibody include (i) an Fab fragment consisting of the VL, VH, CL andCH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains;(iii) an Fv fragment consisting of the VL and VH domains of a single armof an antibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546,1989) which consists of a VH domain; and (v) an F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region. Furthermore, although the two domains of theFv fragment are coded for by separate genes, a synthetic linker can bemade that enables them to be made as a single protein chain (known assingle chain Fv (scFv); (Bird et al., Science 242:423-426, 1988; andHuston et al., Proc. Natl. Acad. Sci. 85:5879-5883, 1988) by recombinantmethods. Such single chain antibodies, as well as dsFv, a disulfidestabilized Fv (Bera et al., J. Mol. Biol. 281:475-483, 1998), anddimeric Fvs (diabodies), that are generated by pairing differentpolypeptide chains (Holliger et al., Proc. Natl. Acad. Sci.90:6444-6448, 1993), are also included.

In one embodiment, antibody fragments for use in this disclosure arethose which are capable of cross-linking their target antigen, forexample, bivalent fragments such as F(ab′)₂ fragments. Alternatively, anantibody fragment which does not itself cross-link its target antigen(for example, a Fab fragment) can be used in conjunction with asecondary antibody which serves to cross-link the antibody fragment,thereby cross-linking the target antigen. Antibodies can be fragmentedusing conventional techniques and the fragments screened for utility inthe same manner as described for whole antibodies. An antibody isfurther intended to include humanized monoclonal molecules thatspecifically bind the target antigen.

“Specifically binds” refers to the ability of individual antibodies tospecifically immunoreact with an antigen. This binding is a non-randombinding reaction between an antibody molecule and the antigen. In oneembodiment, the antigen is TAG-72. Binding specificity is typicallydetermined from the reference point of the ability of the antibody todifferentially bind the antigen of interest and an unrelated antigen,and therefore distinguish between two different antigens, particularlywhere the two antigens have unique epitopes. An antibody thatspecifically binds to a particular epitope is referred to as a “specificantibody.”

In one embodiment the antigen is tumor-associated glycoprotein (TAG-72).Monoclonal, and humanized immunoglobulins are encompassed by thedisclosure. In one example, a murine monoclonal antibody that recognizesthe TAG-72 antigen is CC49. In another example, a humanized CC49antibody is HuCC49. In other examples, a humanized CC49 antibodyincludes a light chain from HuCC49V10 variants V35 or V37, and/or aheavy chain from HuCC49V10 variants V40 or V41. In several examples,variant humanized CC49 antibodies are HuCC49V48 (“V48”), HuCC49V47(“V47”), HuCC49V58 (“V58”) or HuCC49V59 (“V59”). The disclosure alsoincludes synthetic and genetically engineered variants of theseimmunoglobulins.

Antigen: Any molecule that can bind specifically with an antibody. Anantigen is also a substance that evokes immune response, includingproduction of antibodies. Antigens are often foreign substances such asallergens, bacteria or viruses that invade the body. A specific,non-limiting example of an antigen is TAG-72.

CC49 monoclonal antibody: A murine monoclonal antibody of the IgG₁isotype that specifically binds TAG-72 (deposited as ATCC Accession No.HB 9459). This monoclonal antibody is a second generation monoclonalantibody prepared by immunizing mice with TAG-72 that was purified usingthe first generation antibody B72.3 (Colcher et al., Proc. Natl. Acad.Sci. USA 78:3199-3203, 1981). The murine CC49 (mCC49) monoclonalantibody efficiently targets human colon carcinoma xenografts in athymicmice and reduces or eliminates their growth (Colcher et al., Cancer Res.48:4597-4603, 1988). Radiolabeled CC49 has been shown to successfullytarget a number of human tumors including adenocarcinoma, colorectal,breast, prostate and ovarian (Liu et al., Cancer Biother Radiopharm.12:79-87, 1997; Macey et al., Clin. Cancer Res. 3:1547-1555, 1997;Meredith et al., J. Nucl. Med., 37:1491-1496, 1996.)

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chimeric antibody: An antibody which includes sequences derived from twodifferent antibodies, which typically are of different species. Mosttypically, chimeric antibodies include human and murine antibodydomains, generally human constant regions and murine variable regions,murine CDRs and/or murine SDRs.

Complementarity Determining Region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively. By definition, the CDRs of the light chainare bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56(L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded bythe residues at positions 31 and 35b (H-CDR1), 50 and 65 (H-CDR2), 95and 102 (H-CDR3), using the numbering convention delineated by Kabat etal., (1991) Sequences of Proteins of Immunological Interest, 5^(th)Edition, Department of Health and Human Services, Public Health Service,National Institutes of Health, Bethesda (NIH Publication No. 91-3242).

Constant Region: The portion of the antibody molecule which conferseffector functions. In the present disclosure, the variant antibodiesinclude constant regions derived from human immunoglobulins. The heavychain constant region can be selected from any of five isotypes: alpha,delta, epsilon, gamma or mu. Heavy chains of various subclasses (such asthe IgG subclass of heavy chains) are responsible for different effectorfunctions. Thus, by choosing the desired heavy chain constant region,humanized antibodies with the desired effector function can be produced.The light chain constant region can be of the kappa or lambda type.

Cytotoxin: An agent that is toxic for cells. Examples of cytotoxinsinclude radioactive isotopes, chemotherapeutic drugs, bacterial toxins,viral toxins, and proteins contained in venom (for example, insect,reptile, or amphibian venom). A cytokine, such as interleukin-2 orinterferon, can also be a cytotoxin.

Diagnostically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject or tissue beingdiagnosed. For instance, this can be the amount necessary to detect thepresence of a tumor. In one embodiment, a diagnostically effectiveamount is the amount necessary to detect a tumor expressing TAG-72. Whenadministered to a subject, a dosage will generally be used that willachieve target tissue concentrations (for example, in tumors) that hasbeen shown to achieve a desired in vitro effect.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer whichconstitutes the genetic material of most living organisms (some viruseshave genes composed of ribonucleic acid (RNA)). The repeating units inDNA polymers are four different nucleotides, each of which contains oneof the four bases, adenine, guanine, cytosine and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequence of three nucleotides in the mRNA that istranscribed from the DNA.

Effector Molecule: Therapeutic, diagnostic or detection moieties linkedto an antibody, using any number of means known to those of skill in theart. Both covalent and noncovalent linkage means may be used. Theprocedure for linking an effector molecule to an antibody variesaccording to the chemical structure of the effector. Polypeptidestypically contain a variety of functional groups; for example,carboxylic acid (COOH), free amino (—NH₂) or sulfhydryl (—SH) groups,which are available for reaction with a suitable functional group on anantibody to result in the linkage of the effector molecule.Alternatively, the antibody is derivatized to expose or link additionalreactive functional groups. The derivatization may involve linkage ofany of a number of linker molecules such as those available from PierceChemical Company, Rockford, Ill. The linker can be any molecule used tojoin the antibody to the effector molecule. The linker is capable offorming covalent bonds to both the antibody and to the effectormolecule. Suitable linkers are well known to those of skill in the artand include, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (forexample, through a disulfide linkage to cysteine) or to the alpha carbonamino and carboxyl groups of the terminal amino acids.

An “immunoconjugate” is a covalent linkage of an effector molecule, suchas a toxin, a chemical compound, or a detectable label, to an antibody.Specific, non-limiting examples of toxins include, but are not limitedto, abrin, ricin, Pseudomonas exotoxin (such as PE35, PE37, PE38, andPE40), diphtheria toxin, anthrax toxin, botulinum toxin, or modifiedtoxins thereof. For example, Pseudomonas exotoxin and diphtheria toxinare highly toxic compounds that typically bring about death throughliver toxicity. Pseudomonas exotoxin and diphtheria toxin, however, canbe modified into a form for use as an immunotoxin by removing the nativetargeting component of the toxin (for example, domain Ia of Pseudomonasexotoxin and the B chain of diphtheria toxin) and replacing it with adifferent targeting moiety, such as an antibody. Other toxic agents,that directly or indirectly inhibit cell growth or kill cells, includechemotherapeutic drugs, cytokines, for example interleukin (IL)-2, IL-4,IL-10, tumor necrosis factor-alpha, or interferon-gamma, radioactiveisotopes, viral toxins, or proteins contained within, for example,insect, reptile, or amphibian venom. Specific, non-limiting examples ofdetectable labels include, but are not limited to, radioactive isotopes,enzyme substrates, co-factors, ligands, chemiluminescent agents,fluorescent agents, haptens, or enzymes.

In one embodiment, an antibody is joined to an effector molecule. Inanother embodiment, an antibody joined to an effector molecule isfurther joined to a lipid or other molecule to a protein or peptide toincrease its half-life in the antibody. The linkage can be, for example,either by chemical or recombinant means. In one embodiment, the linkageis chemical, wherein a reaction between the antibody moiety and theeffector molecule has produced a covalent bond formed between the twomolecules to form one molecule. A peptide linker (short peptidesequence) can optionally be included between the antibody and theeffector molecule.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (forexample, when exposed to tumor-associated enzymes or acidic pH) may beused.

In view of the large number of methods that have been reported forlinking a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (for example, enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for linking a given agent to anantibody.

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Epitope: A site on an antigen recognized by an antibody, as determinedby the specificity of the antibody amino acid sequence. Epitopes arealso called antigenic determinants.

Framework Region: Amino acid sequences interposed between CDRs. Antibodyframework region includes variable light and variable heavy frameworkregions. The framework regions serve to hold the CDRs in an appropriateorientation for antigen binding. The numbering of the residues in thelight chain and heavy chain framework regions follows the numberingconvention delineated by Kabat et al., (1991, supra).

High binding affinity: Affinity of an antibody for an antigen where therelative affinity of the humanized CC49 antibody is similar or increasedas compared to that of a parent CC49 antibody, for example HuCC49V10. Inone embodiment, affinity is calculated by a modification of theScatchard method described by Frankel et al., Mol. Inmunol., 16:101-106,1979. One of skill in the art can readily identify a statistical testthat determines a statistically significant result, for example, theStudent's t-test, the Wilcoxon two sample test, or the Median test. Inone embodiment, the humanized CC49 antibody has a high binding affinityfor TAG-72 that is at least about 0.6×10⁻⁸ M. In other embodiments, thehumanized CC49 antibody has a high binding affinity for TAG-72 that isat least about 1.0×10⁻⁸, about 1.2×10⁻⁸, about 1.5×10⁻⁸, about 2.0×10⁻⁸, about 2.5×10⁻⁸, about 3.0×10⁻⁸, about 3.5×10⁻⁸, about 4.0×10⁻⁸, about4.5×10⁻⁸, or about 5.0×10⁻⁸ M.

In another embodiment, a high binding affinity is measured by anantigen/antibody dissociation rate of a humanized CC49 antibody that issignificantly lower than the parent antibody. In yet another embodiment,a high binding affinity is measured by a competition radioimmunoassay,where the amount of antibody needed for 50% inhibition of the binding of¹²⁵I-labeled HuCC49 antibody to BSM is less than that required by theparent CC49 antibody. In another embodiment, a high binding affinity ismeasured by flow cytometry as an increased number of gated cells labeledwith humanized CC49 antibody compared to the number of cells labeled bythe parent CC49 antibody.

HAMA (Human anti-murine antibody) response: An immune response in ahuman subject to the variable and constant regions of a murine antibodythat has been administered to the subject. Repeated antibodyadministration may lead to an increased rate of clearance of theantibody from the patient's serum and may also elicit allergic reactionsin the subject.

Humanized antibody: A human antibody genetically engineered to includemouse hypervariable regions, CDRs and/or SDRs. In one embodiment, theDNA encoding hypervariable loops of mouse monoclonal antibodies orvariable regions selected in phage display libraries is inserted intothe framework regions of human Ig genes. Antibodies can be “customized”to have a desired binding affinity or to be minimally immunogenic in thehumans treated with them.

Humanized CC49 antibodies: CC49 antibodies humanized by grafting CC49CDRs Kashmiri et al., Hybridoma, 14: 461-473, 1995) or SDRs (Tamura etal., J. Immunol. 164:1432-1441, 2000; WO 00/26394) onto the frameworksof the relevant human antibodies. CC49 CDRs include synthetic amino acidsequences that are identical in sequence to the CC49 CDRs. CC49 can behumanized by graffing only CC49 CDRs that are important for antigenbinding onto the variable light and variable heavy framework regions of,for example, LEN and 21/28′CL human antibodies and additionallyreplacing non-specificity determining residues (SDRs) in the murine CDRswith the corresponding residue in the human antibody. A limited numberof murine framework residues, can be included in a humanized antibody.In one embodiment, no murine resides are included in the frameworkregion. In other embodiments, at most about one, two, three four, five,six, seven, eight, nine, ten, twelve, fourteen, fifteen, or sixteenmurine amino acids are included in the human framework. A specifichumanized CC49 antibody, termed HuCC49 has been deposited with ATCC asHB-12404.

The variant HuCC49V10 carries the L-CDR-1 and L-CDR2 of the humanantibody LEN, and a threonine at position 97 in the CC49 L-CDR3 isreplaced with a serine residue present at the corresponding position inthe human antibody LEN. The variant HuCC49V10 also has severalsubstitutions in the heavy chain. Specifically, an asparagine atposition 60 in the murine CC49 H-CDR2 is replaced with a serine, aglutamic acid at position 61 in the murine CC49 H-CDR2 is replaced witha glutamine, an arginine at position 62 in the murine CC49 H-CDR2 isreplaced with a lysine, and a lysine at position 64 in the murine CC49H-CDR2 is replaced with a glutamine. It should be noted that HuCC49V10is described in U.S. patent application Ser. No. 09/830,748 and PCTPublication No. WO 00/26394, both of which are incorporated herein byreference. HuCC49V10 was deposited with ATCC on Aug. 28, 2003, and hasATCC Accession No. PTA-5416. For the purposes of this disclosure,HuCC49V10 can be referred to as the parental antibody.

Additional humanized CC49 antibodies are HuCC49V10-14 (ATCC AccessionNo. PTA-4182, also termed HuCC49V14) and HuCC49V10-15 (ATCC AccessionNo. PTA-4183, also termed HuCC49V15).

Other specific, non-limiting examples of a humanized CC49 monoclonalantibody are HuCC49V10 variants V35, V37, V40, V41, V47, V48, V58, andV59. Antibody V59 was deposited with ATCC on Aug. 28, 2003, and has ATCCAccession No. PTA-5415. Methods for making these antibodies, and theamino acid sequence of the VL and VH chains of these antibodies areprovided herein.

Idiotype: The property of a group of antibodies or T cell receptorsdefined by their sharing a particular idiotope (an antigenic determinanton the variable region); for instance, antibodies that share aparticular idiotope belong to the same idiotype. “Idiotype” may be usedto describe the collection of idiotopes expressed by an Ig molecule. An“anti-idiotype” antibody may be prepared to a monoclonal antibody bymethods known to those of skill in the art and may be used to preparepharmaceutical compositions.

Immune cell: Any cell involved in a host defense mechanism. These caninclude, for example, T cells, B cells, natural killer cells,neutrophils, mast cells, macrophages, antigen-presenting cells,basophils, eosinophils, and neutrophils.

Immune response: A response of a cell of the immune system, such as aneutrophil, a B cell, or a T cell, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In another embodiment, the response is against an antibody,such as a HAMA response, including an anti-variable region response.

Immunogenicity: A measure of the ability of a targeting protein ortherapeutic moiety to elicit an immune response (humoral or cellular)when administered to a subject.

In one example, a variant, such as, but not limited to, V59, has minimalimmunogenicity (compared to the parental HuCC49V10 antibody). In oneexample a reduced or minimal immunogenicity, as compared to a parentalantibody, is an IC₅₀ value for serum EA is at least about a 2-fold,5-fold, 1 0-fold, 20-fold, 25-fold, 30-fold, or 35-fold higher than thatof a parental antibody. However, other assays can be used to measureimmunogenicity.

Immunoreactivity: A measure of the ability of an Ig to recognize andbind to a specific antigen.

Isolated: An biological component (such as a nucleic acid, peptide orprotein) that has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, for instance, otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins that have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule to facilitate detection of thatmolecule. Specific, non-limiting examples of labels include fluorescenttags, chemiluminescent tags, haptens, enzymatic linkages, andradioactive isotopes.

Ligand contact residue or Specificity Determining Residue (SDR): Aresidue within a CDR that is involved in contact with a ligand orantigen. A ligand contact residue is also known as a specificitydetermining residue (SDR). A non-ligand contact residue is a residue ina CDR that does not contact a ligand. A non-ligand contact residue canalso be a framework residue.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Minimally immunogenic: An antibody that generates a reduced, for examplelow, immune response when administered to a subject, such as a humansubject. In one embodiment, immunogenicity is measured in a competitivebinding assay. In one specific, non-limiting example, immunogenicity isthe ability of a variant HuCC49 antibody to prevent a parental HuCC49V10antibody from binding to CC49 anti-idiotypic antibodies in a patient'sserum. For example, if a variant HuCC49 antibody competes with an equalmolar amount of the parental HuCC49V10 antibody (i.e. elicits greaterthan about 50% inhibition of parental HuCC49V10 binding toanti-idiotypic antibodies in a patient's serum) then the variant HuCC49antibody is immunogenic. In another example, if a variant HuCC49antibody competes poorly with an equal molar or less amount of theparental HuCC49V10 antibody (i.e. elicits about 50% or less inhibitionof parental HuCC49V10 binding to anti-idiotypic antibodies in apatient's serum) then the variant HuCC49 antibody is minimallyimmunogenic. In another embodiment, if a five-fold or greater molarconcentration of a variant HuCC49 antibody is required to achieve about50% inhibition of binding of the parental antibody to its cognateanti-idiotypic antibodies present in a subject's sera, then the variantantibody is minimally immunogenic.

Monoclonal antibody: An antibody produced by a single clone ofB-lymphocytes. Monoclonal antibodies are produced by methods known tothose of skill in the art, for instance by making hybridantibody-forming cells from a fusion of myeloma cells with immune spleencells.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Oligonucleotide: A linear single-stranded polynucleotide sequence of upto about 200 nucleotide bases in length, for example a polymer ofdeoxyribonucleotides or ribonucleotides which is at least 6 nucleotides,for example at least 15, 50, 100 or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell.

A “therapeutically effective amount” is a quantity of a specificsubstance sufficient to achieve a desired effect in a subject beingtreated. For instance, this can be the amount necessary to inhibit orsuppress growth of a tumor or to decrease a sign or symptom of the tumorin the subject. In one embodiment, a therapeutically effective amount isthe amount necessary to eliminate a tumor. When administered to asubject, a dosage will generally be used that will achieve target tissueconcentrations (for example, in tumors) that has been shown to achieve adesired in vitro effect.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15^(th) Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of humanized CC49 monoclonalantibodies disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: A single-stranded linear nucleotide sequence, includingsequences of greater than 100 nucleotide bases in length.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred in nature. The termpolypeptide or protein as used herein encompasses any amino acidsequence and includes, but may not be limited to, modified sequencessuch as glycoproteins. The term polypeptide is specifically intended tocover naturally occurring proteins, as well as those that arerecombinantly or synthetically produced.

Substantially purified polypeptide as used herein refers to apolypeptide that is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

A non-conservative amino acid substitution can result from changes in:(a) the structure of the amino acid backbone in the area of thesubstitution; (b) the charge or hydrophobicity of the amino acid; or (c)the bulk of an amino acid side chain. Substitutions generally expectedto produce the greatest changes in protein properties are those inwhich: (a) a hydrophilic residue is substituted for (or by) ahydrophobic residue; (b) a proline is substituted for (or by) any otherresidue; (c) a residue having a bulky side chain, for example,phenylalanine, is substituted for (or by) one not having a side chain,for example, glycine; or (d) a residue having an electropositive sidechain, for example, lysyl, arginyl, or histadyl, is substituted for (orby) an electronegative residue, for example, glutamyl or aspartyl.

Variant amino acid sequences can be, for example, be 80%, 90% or even95% or 98% identical to the native amino acid sequence. Programs andalgorithms for determining percentage identity can be found at the NCBIwebsite.

Preventing or treating a disease: Preventing a disease refers toinhibiting completely or in part the development or progression of adisease, for example in a person who is known to have a predispositionto a disease, such as colorectal cancer, breast, ovarian, or prostatecancer. An example of a person with a known predisposition is someonewith a history of cancer in the family, or who has been exposed tofactors that predispose the subject to the development of a tumor.Treating a disease refers to a therapeutic intervention that inhibits,or suppresses the growth of a tumor, eliminates a tumor, ameliorates atleast one sign or symptom of a disease or pathological condition, orinterferes with a pathophysiological process, after the disease orpathological condition has begun to develop.

Protein: A biological molecule encoded by a gene and comprised of aminoacids.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or was made artificially. Artificialcombination is often accomplished by chemical synthesis or, morecommonly, by the artificial manipulation of isolated segments of nucleicacids, for example, by genetic engineering techniques. Similarly, arecombinant protein is one encoded by a recombinant nucleic acidmolecule.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency, will vary depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (for example, GC v. ATcontent), and nucleic acid type (for example, RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific, non-limiting example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). One of skill in the art can readily determine variations onthese conditions (for example, Molecular Cloning: A Laboratory Manual,2^(nd) ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001). Washing can be carried out usingonly one of these conditions, for example, high stringency conditions,or each of the conditions can be used, for example, for 10-15 minuteseach, in the order listed above, repeating any or all of the stepslisted. However, as mentioned above, optimal conditions will vary,depending on the particular hybridization reaction involved, and can bedetermined empirically.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

TAG (Tumor-Associated Glycoprotein)-72: A cell-surface glycoprotein thatis expressed on human carcinomas, including adenocarcinoma, colorectal,gastric, pancreatic, breast, lung and ovarian carcinomas. TAG-72 has ahigh molecular weight (greater than 1'10⁶) as measured by size-exclusionchromatography, a density of 1.45 g/ml, is resistant to Chondroitinasedigestion, expresses blood group-related oligosaccharides, and isheavily sialylated with O-glycosidically linked oligosaccharidescharacteristic of mucins. These characteristics suggest that TAG-72 is amucin-like molecule (Johnson et al., Cancer Res. 46:850-857, 1986,incorporated herein by reference).

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate a tumor. When administered to a subject, adosage will generally be used that will achieve target tissueconcentrations (for example, in tumors) that has been shown to achieve adesired in vitro effect.

Treatment: Refers to both prophylactic inhibition of initial infectionor disease, and therapeutic interventions to alter the natural course ofan untreated infection or disease process, such as a tumor growth or aninfection with a bacteria.

Tumor: A neoplasm that may be either malignant or non-malignant. Tumorsof the same tissue type are primary tumors originating in a particularorgan (such as breast, prostate, bladder or lung). Tumors of the sametissue type may be divided into tumor of different sub-types (a classicexample being bronchogenic carcinomas (lung tumors) which can be anadenocarcinoma, small cell, squamous cell, or large cell tumor). Breastcancers can be divided histologically into scirrhous, infiltrative,papillary, ductal, medullary and lobular. In one embodiment, cells in atumor express TAG-72.

Variable region (also variable domain or V domain): The regions of boththe light chain and the heavy chain of an Ig that containantigen-binding sites. The regions are composed of polypeptide chainscontaining four relatively invariant “framework regions” (FRs) and threehighly variant “hypervariable regions” (HVs). Because the HVs constitutethe binding site for antigen(s) and determine specificity by forming asurface complementarity to the antigen, they are more commonly termedthe “complementarity-determining regions,” or CDRs, and are denotedCDR1, CDR2, and CDR3. Because both of the CDRs from the heavy and lightchain domains contribute to the antigen-binding site, it is thethree-dimensional configuration of the heavy and the light chains thatdetermines the final antigen specificity.

Within the heavy and light chain, the framework regions surround theCDRs. Proceeding from the N-terminus of a heavy or light chain, theorder of regions is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As used herein, theterm “variable region” is intended to encompass a complete set of fourframework regions and three complementarity-determining regions. Thus, asequence encoding a “variable region” would provide the sequence of acomplete set of four framework regions and threecomplementarity-determining regions.

Variant humanized CC49 antibody: A humanized CC49 antibody that has atleast one amino acid substitution of a murine residue, and specificallybinds TAG-72. A humanized CC49 antibody can have at most 2, at most 3,at most 4, at most 5, at most 7, at most 9, at most 10, at most 12, atmost 14, at most 16, or more amino acid substitutions. In oneembodiment, a variant humanized CC49 antibody is a variant of HuCC49V10.Specific non-limiting examples of variant humanized CC49 antibodiesinclude V35, V37, V40, V41, V47, V48, V58, and V59.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless the context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Minimally Immunogenic Variants of Humanized CC49 V10

The majority of patients treated with murine CC49 (mCC49) monoclonalantibody generate HAMA responses (Divgi et al., J Nucl Med 36:586-592,1995; Divgi et al., Clin Cancer Res 1:1503-1510, 1995; Macey et al.,Clin Cancer Res 3:1547-1555, 1997; Rucker et al., J Immunother 22:80-4,1999; Slovin et al., Clin Cancer Res 4:643-651, 1998; Tempero et al., JClin Oncol 15:1518-1528, 1997), preventing repeated administration ofthe monoclonal antibody. To circumvent this problem, a humanized CC49(HuCC49) was developed by the conventional approach of grafting all six(three heavy chain and three light chain) mCC49 CDRs onto the VL and VHframeworks of the human Abs LEN and 21/28′CL, respectively, whileretaining murine framework residues that may be required for theintegrity of the antigen combining site structure (FIG. 9; Kashmiri etal., Hybridoma, 14:461-473, 1995; see also U.S. Pat. No. 6,495,137,incorporated herein by reference). The resulting HuCC49, which shows noevidence of patients' antibody response after one injection (Forero etal., Cancer Biotherapy & Radiopharmaceuticals, 2002), retains itsspecificity to TAG-72, showing only 2- to 3-fold lower affinity thanthat of mCC49. HuCC49 was deposited with ATCC and has ATCC Accession No.HB-12404. However, an immune response to the variable (V) region canstill develop.

To minimize any possible anti-V region antibody response that couldstill be evoked in patients by HuCC49, mCC49 was humanized by SDRgrafting (Tamura et al., J Immunol 164:1432-1441,2000). In theSDR-grafted HuCC49V10 (V10), referred to as the parental antibody inthis disclosure, the light chain L-CDR1 and L-CDR2 of CC49 were replacedwith their counterparts from the human antibody LEN. The murine residueat position 97 of L-CDR3 (threonine) also was replaced with the residuelocated at the corresponding position in the human antibody (serine).The variant HuCC49V10 also has several substitutions in the heavy chain.Specifically, an asparagine at position 60 in the murine CC49 H-CDR2 isreplaced with a serine, a glutamic acid at position 61 in the murineCC49 H-CDR2 is replaced with a glutamine, an arginine at position 62 inthe murine CC49 H-CDR2 is replaced with a lysine, and a lysine atposition 64 in the murine CC49 H-CDR2 is replaced with a glutamine.These substitutions are shown in Table I, below. Murine frameworkresidues that were presumed to be indispensable for maintaining theintegrity of the antibody-combining site were retained during thegeneration of V10. In HuCC49V10, murine CC49 framework residues wereretained at positions 5, 19, 21, 43, 78, 100 and 106 of the light chainand at positions 12, 20, 38, 40, 48, 66, 67, 69, 71, 80, 91 and 93 ofthe heavy chain (following the numbering convention delineated by Kabatet al., 1991, supra). Compared to the CDR-grafted HuCC49, the V10antibody shows only a 2- to 3-fold reduction in its binding affinity tothe TAG-72 antigen.

In vitro sera reactivity studies reveal that compared to HuCC49, V10shows a dramatic decrease in its reactivity to the anti-V region Abspresent in the sera of patients who were administered 177 Lu-labeledmCC49 in a phase I clinical trial (Gonzales et al., J Immunol Methods268:197-210, 2002; Tamura et al., J Immunol 164:1432-1441, 2000). Usinga surface plasmon resonance (SPR)-based competition assay to measuresera reactivity, it has been shown that compared to HuCC49, V10 displays10- to 300-fold lower reactivity to the sera of several patients(Gonzales et al., J Immunol Methods 268:197-210, 2002). Although SDRgrafting of mCC49 has rendered V10 minimally reactive to sera frompatients who had earlier been administered mCC49 in clinical trials, therecipient of V10 can still elicit an anti-V region response against thepotentially immunogenic murine framework residues that were retained fortheir presumed indispensability in maintaining the integrity of the Abcombining site (Kashmiri et al., Hybridoma 14:461-473, 1995; Tamura etal., J Immunol 164:1432-1441, 2000). It should be noted that HuCC49V10is described in U.S. patent application Ser. No. 09/830,748 and PCTPublication No. WO 00/26394, both of which are incorporated herein byreference. These documents also disclose the amino acid sequence of LENand 21/28′CL. HuCC49V10 was deposited with ATCC and has ATCC AccessionNo. PTA-5416.

The antibodies disclosed herein include the CDRs from HuCC49V10 (shownin Table I, below, and in FIG. 9) in a human framework. However, theantibody can also include a non-conservative substitution at position 91of HuCC49V10 LCDR3 (SEQ ID NO: 11), such as a tyrosine to prolinesubstitution (HuCC49V10-14; see U.S. patent application Ser. No.60/393,077 and PCT Patent Application No. PCT/US03/20367, filed Jun. 26,2003, both of which are incorporated herein by reference).Alternatively, the antibody can have a leucine at position 27b ofHuCC49V10 LCDR1 (SEQ ID NO: 9) and a non conservative amino acidsubstitution at position 91 of HuCC49V10 LCDR3 (SEQ ID NO: 11), such asa proline at position 91 (HuCC49V10-15; see U.S. patent application Ser.No. 60/393,077 and PCT Patent Application No. PCT/US03/20367, filed Jun.26, 2003, both of which are incorporated herein by reference. TABLE ILight and Heavy Chain CDR Sequences of HuCC49 and HuCC49V10 Light chainCDR1 24¹ 25 26 27 a b c d² e f 28 29 30 31 32 33 34 HuCC49 Lys Ser SerGln Ser Leu Leu Tyr Ser Gly Asn Gln Lys Asn Tyr Leu Ala (SEQ ID NO: 31)V10³ Lys Ser Ser Gln Ser VAL ⁴ Leu Tyr Ser SER Asn SER Lys Asn Tyr LeuAla (SEQ ID NO: 9) CDR2 50 51 52 53 54 55 56 HuCC49 Trp Ala Ser Ala ArgGlu Ser (SEQ ID NO: 32) V10 Trp Ala Ser THR Arg Glu Ser (SEQ ID NO: 10)CDR3 89 90 91 92 93 94 95 96 97 HuCC49 Gln Gln Tyr Tyr Ser Tyr Pro LeuThr (SEQ ID NO: 33) V10 Gln Gln Tyr Tyr Ser Tyr Pro Leu SER (SEQ ID NO:11) Heavy chain CDR1 31 32 33 34 35 HuCC49 Asp His Ala Ile His (SEQ IDNO: 34) V10 Asp His Ala Ile His (SEQ ID NO: 12) CDR2 50 51 52 a 53 54 5556 57 58 59 60 61 62 63 64 65 HuCC49 Tyr Phe Ser Pro Gly Asn Asp Asp PheLys Tyr Asn Glu Arg Phe Lys Gly (SEQ ID NO: 35) V10 Tyr Phe Ser Pro GlyAsn Asp Asp Phe Lye Tyr SER GLN LYS Phe GLN Gly (SEQ ID NO: 13) CDR3 9596 97 98 99 100 a b 101 102 HuCC49 Set Leu Asn Met Ala — — — — Tyr (SEQID NO: 36) V10 Set Leu Asn Met Ala Tyr (SEQ ID NO: 14)¹Amino acid residues are numbered as described by Kabat.²Residue position shown in BOLD denote the specificity-determiningresidues (SDRs).³V10 represents HuCC49V10⁴HuCC49V10 residues derived from corresponding positions of human MAbsLEN and 21/28′CL shown in ALL CAPS

Thus, in one embodiment, the antibody does not include L-CDR3 fromHuCC49V10, but includes L-CDR3 from HuCC49V14 (ATCC Accession No.PTA-4182). In this embodiment the L-CDR3, or SDR from HuCC49V14 L-CDR3,can be utilized. Thus, the antibodies include an L-CDR3 (or SDR fromL-CDR3) from HuCC49V14, and a L-CDR1, a L-CDR2, a H-CDR-1, a H-CDR2, anda H-CDR3 from humanized antibody HuCC49V10. The L-CDR1, L-CDR2, L-CDR3are within a HuCC49V10 light chain framework region that includes thecorresponding amino acid from LEN at position 5, 19, 21, and 106 in thelight chain. The H-CDR1, H-CDR2, and H-CDR3 are within a heavy chainHuCC49V10 framework comprising a corresponding human 21/28′ CL residueat positions 20, 38,48, 66, 67, 69, and 80 in the heavy chain. In oneembodiment, corresponding murine CC49 framework residues are retained atpositions 71, 91, 93 of the heavy chain. These humanized CC49 antibodiesretain binding affinity for TAG-72 and have reduced immunogenicity, ascompared to a parental HuCC49V10 antibody.

The L-CDRs from HuCC49V15 (see ATCC Accession No. PTA-4183) can also beutilized. In this embodiment, the antibody does not include L-CDR1 orL-CDR3 (or an SDR from L-CDR1 or L-CDR3) from HuCC49V10, but includesL-CDR1 and L-CDR3 (or an SDR from L-CDR1 or L-CDR3) from HuCC49V15. Theantibody also includes a L-CDR2, and a HCDR1, a H-CDR2, and a H-CDR3from humanized antibody HuCC49V10. The L-CDR1, L-CDR2, L-CDR3 are withina HuCC49V10 light chain framework region that includes the correspondingframework amino acid from LEN at position 5, 19, 21, and 106 in thelight chain.

The H-CDR1, H-CDR2, and H-CDR3 are within a heavy chain HuCC49V10framework comprising a corresponding framework human 21/28′ CL residueat positions 20, 38, 48, 66, 67, 69, and 80 in the heavy chain. In oneembodiment, corresponding murine CC49 framework residues are retained atpositions 71, 91, 93 of the heavy chain. These humanized CC49 antibodiesretain binding affinity for TAG-72 and have reduced immunogenicity, ascompared to a parental HuCC49V10 antibody. HuCC49V14 and HuCC49V15 havebeen previously disclosed (see U.S. patent application Ser. No.60/393,077 and PCT Patent Application No. PCT/US03/20367, filed Jun. 26,2003, both of which are incorporated herein by reference).

The light chain frameworks of human mAb LEN have the followingsequences: (SEQ ID NO: 21) FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 22)FR2: WYQQKPGQPPLLIY (SEQ ID NO: 23) FR3:GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 24) FR4: FGQGQTKLEIK

The heavy chain frameworks of human mAb 21/28′ CL have the followingsequences: (SEQ ID NO: 26) FR1: QVQLVQSGAEVKKPQASVKVSCKASQYTFT (SEQ IDNO: 27) FR2: WVRQAPGQRLEWMG (SEQ ID NO: 28) FR3:RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 29) FR4: WGQGTLVTVSS.

The light chain framework regions of HuCC49V10 are listed below: (SEQ IDNO: 1) FR1: DIVMSQSPDSLAVSLGERVTLNC (SEQ ID NO: 2) FR2: WYQQKIPGQSPKLLIY(SEQ ID NO: 3) FR3: GVPDRFSGSGSGTDFTLTISSVQAEDVAVYYC (SEQ ID NO: 4) FR4:FGAGTKLELK

The heavy chain framework regions of HuCC49V10 are listed below: (SEQ IDNO: 5) FR1: QVQLVQSGAEVVKPGASVKISCKASGYTFT (SEQ ID NO: 6) FR2:WVKQNPGQRLEWIG (SEQ ID NO: 7) FR3: KATLTADTSASTAYVELSSLRSEDTAVYFCTR (SEQID NO: 8) FR4: WGQGTLVTVSS

The antibodies disclosed herein bind TAG-72 and include the amino acidsequence of the CDRs of HuCC49V10, but include substitutions of murineresidues retained in the framework of the HuCC49V10 light chain or heavychain. Thus, the variant antibodies disclosed herein include a residuefrom the corresponding position in a parental human antibody atspecified positions (these are specified herein using the numberingconvention delineated by Kabat et al., 1991, supra). Murine frameworkresidues retained in the light chain of HuCC49V10 are at positions 5,19, 21, 43, 78, 100 and 106. In one embodiment, the variant HuCC49V10antibody includes a substitution of residues 5, 19, 21 and 106 in theHuCC49V10 framework of the light chain with corresponding human residuesfrom LEN. In one example, the antibody includes a substitution atpositions 5, 19, 21, 78 and 106 (see FIG. 2, frameworks 1-4 of variantHuCC49V10 antibody V35; SEQ ID NOs: 37-40) of the framework of the lightchain. In another example, the antibody includes a substitution atpositions 5, 19, 21, 43, 78, 100 and 106 of the framework of the lightchain (see FIG. 2, frameworks 1-4 of variant HuCC49V10 antibody V37; SEQID NOs: 41-44). The corresponding residues from LEN are shown in FIG. 9.

Murine framework residues retained in the heavy chain of HuCC49V10 areat positions 12, 20, 38, 40, 48, 66, 67, 69, 71, 80, 91, and 93(following the numbering convention delineated by Kabat et al., 1991,supra). In one embodiment, the variant HuCC49V10 antibody includessubstitutions of residues at positions 20, 38, 48, 66, 67, 69, and 80 inthe heavy chain HuCC49V10 framework with corresponding human residuesfrom 21/28′CL. In one example, the heavy chain framework includes thecorresponding human residues at positions 20, 38, 48, 66, 67, 69, 80(see FIG. 2, frameworks 1-4 of HuCC49V10 variant V40; SEQ ID NOs:45-48). In another example, the heavy chain framework includescorresponding human amino acid residues at positions number 12, 20, 38,40, 48, 66, 67, 69, 80 of the heavy chain framework (see FIG. 2,frameworks 1-4 of HuCC49V10 variant V41; SEQ ID NOs: 49-52).

One of skill in the art can readily design antibodies including one ofthe light chain framework regions of the variants disclosed herein (V35and V37) and one of the heavy chain framework regions disclosed herein(V40 and V10), wherein the antibody includes the CDRs and/or SDRs froman antibody that specifically binds TAG-72, such as HuCC49V10. Specificexamples of antibodies of use are disclosed herein, namely V47, V48, V58and V59, which include one VL selected from the group consisting of V35and V37, and one VH selected from the group consisting of V40 and V41.

For example, an antibody (V47) can be produced that includes V35 andV40. In another example, an antibody (V48) can be produced that includesV37 and V40. In yet another example, an antibody (V58) can be producedthat includes V35 and V41. In a further example, an antibody (V59) canbe produced that includes V37 and V41. It should be noted that all ofthese antibodies include the CDRs of HuCC49V10.

In one example, the antibody has a VL which includes (1) a light chainframework region including the amino acid sequences set forth as SEQ IDNOs: 41-44 and (2) L-CDRs including the amino acid sequences set forthas SEQ NOs: 9-12. The antibody also includes a VH which includes (1)heavy chain framework region including the amino acid sequences setforth as SEQ ID NOs: 49-52 and (2) H-CDRs including the amino acidsequences set forth as SEQ ID NOs: 12-14. This humanized CC49 antibodyretains binding affinity for TAG-72 and has reduced inununogenicity, ascompared to a parental humanized CC49 V10 antibody.

The sequence information provided herein for the light chain frameworkregions and the heavy chain framework regions disclosed herein can beused to produce additional antibodies, including the LCDRs and theHCDRs, or heavy and light chain SDRs, from any other antibody thatspecifically binds TAG-72. For example, the CDRs or SDRs from HuCC49V14(ATCC Accession No. PTA-4182) can be utilized. The CDRs and SDRs fromHuCC49V15 (ATCC Accession No. PTA-4183) can also be utilized.

The variant antibodies disclosed herein, such as V47, V48, V58 and V59,contain a reduced number of murine residues, and consequently, reducedimmunogenicity, when compared to HuCC49 and HuCC49V10. Without beingbound by theory, it is believed that this is due to a reduced number ofamino acids that correspond to a murine antibody. Nonetheless, thevariants of the invention retain a binding affinity that is similar oris increased as compared to HuCC49V10 . Thus, the humanized monoclonalantibodies disclosed herein bind TAG-72 with high binding affinity. Inone embodiment, the humanized CC49 antibody has a high binding affinityfor TAG-72 that is at least about 0.6×10⁻⁸ M. In other embodiments, thehumanized CC49 antibody has a high binding affinity for TAG-72 that isat least about 1.0×10⁻⁸, about 1.2×10⁻⁸, about 1.5×10⁻⁸, about 2.0×10⁻⁸,about 2.5×10⁻⁸, about 3.0×10⁻⁸, about 3.5×10⁻⁸, about 4.0×10⁻⁸, about4.5×10⁻⁸, or about 5.0×10⁻⁸ M. In one embodiment, the humanized CC49antibody has a high binding affinity if it has a significantly lowerantigen/antibody dissociation rate compared to that of the parentHuCC49V10 antibody. In another embodiment, the humanized CC49 antibodyhas a high binding affinity if less antibody is required for a 50%inhibition of the binding of ¹²⁵I-labeled HuCC49 to BSM compared to theparent HuCC49V10 antibody. In yet another embodiment, the humanized CC49antibody has a high binding affinity when the number of cells labeledwith humanized CC49 antibody is significantly greater than the number ofcells labeled by the parent HuCC49V10 antibody, as measured by flowcytometry.

Immunogenicity of variant HuCC49 antibodies can be measured in acompetitive binding assay as the ability of a variant HuCC49 antibody toprevent a CC49, HuCC49 or HuCC49V10 antibody from binding toanti-idiotypic antibodies in a human subject's serum. In one embodiment,the variant antibody is minimally immunogenic in a subject. In oneembodiment, at least about five-fold higher molar concentration of thevariant humanized CC49 antibody, than that of the parental HuCC49V10antibody, is required to elicit 50% inhibition of the parental HuCC49V10binding to its cognate anti-idiotypic antibodies in a subject's sera. Inother embodiments, at least about ten-fold, at least about twentyfive-fold, at least about fifty-fold, at least about seventy-fold, or atleast about one hundred-fold higher molar concentration of the varianthumanized CC49 antibody, than that of the parental antibody HuCC49V10,is required to elicit 50% inhibition of the parental HuCC49V10 bindingto its cognate anti-idiotypic antibodies in a subject's sera.

Effector molecules, for example, therapeutic, diagnostic, or detectionmoieties, can be linked to a variant humanized CC49 antibody thatspecifically binds TAG-72, using any number of means known to those ofskill in the art. Thus, a variant humanized CC49 antibody with an aminoacid substitution can have any one of a number of different types ofeffector molecules linked to it. In one embodiment, the antibody islinked to a detectable label. In some embodiments, the antibody islinked to a radioactive isotope, an enzyme substrate, a co-factor, aligand, a chemiluminescent agent, a fluorescent agent, a hapten, or anenzyme. In another embodiment, the antibody is linked to a cytotoxin(see below). In other embodiments, the antibody is linked to achemotherapeutic drug, a radioactive isotope, a bacterially-expressedtoxin, a virally-expressed toxin, or a venom protein. In yet otherembodiments, the antibody is linked to a cytokine. Specific,non-limiting examples of cytokines are IL-2, IL-4, IL-10, TNF-alpha andIFN-gamma. The antibody can be linked to an effector molecule by acovalent or non-covalent means.

Polynucleotides encoding the VL and/or the VH of humanized antibodiesthat bind TAG-72, such as V47, V48, V58 and V59 are also provided. Thesepolynucleotides include DNA, cDNA and RNA sequences which encode thehumanized antibody. It is understood that all polynucleotides encodingthese antibodies are also included herein, as long as they encode apolypeptide with the recognized activity, such as the binding to TAG-72.The polynucleotides of this disclosure include sequences that aredegenerate as a result of the genetic code. There are 20 natural aminoacids, most of which are specified by more than one codon. Therefore,all degenerate nucleotide sequences are included as long as the aminoacid sequence of the antibody encoded by the nucleotide sequence isfunctionally unchanged.

Primers, such as PCR primers, can readily be prepared that hybridize toa nucleic acid sequence encoding a specific VH or VL, or a componentthereof. In one embodiment, the primers include at least ten, at least15, 16, 17, 18, 18, or 20 consecutive nucleotides of a nucleic acidencoding the VH or VL of interest. Also included are fragments of theabove-described nucleic acid sequences that are at least 15 bases inlength, which is sufficient to permit the fragment to selectivelyhybridize to DNA that encodes the antibody of interest underphysiological conditions. The term “selectively hybridize” refers tohybridization under moderately or highly stringent conditions, whichexcludes non-related nucleotide sequences.

A nucleic acid encoding a VL and/or VH of a humanized antibody thatspecifically binds TAG-72 can be cloned or amplified by in vitromethods, such as the polymerase chain reaction (PCR), the ligase chainreaction (LCR), the transcription-based amplification system (TAS), theself-sustained sequence replication system (3SR) and the Qβ replicaseamplification system (QB). For example, a polynucleotide encoding theprotein can be isolated by polymerase chain reaction of cDNA usingprimers based on the DNA sequence of the molecule. A wide variety ofcloning and in vitro amplification methodologies are well known topersons skilled in the art. PCR methods are described in, for example,U.S. Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant.Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press,NY, 1989). Polynucleotides also can be isolated by screening genomic orcDNA libraries with probes selected from the sequences of the desiredpolynucleotide under stringent hybridization conditions.

The polynucleotides include a recombinant DNA which is incorporated intoa vector; into an autonomously replicating plasmid or virus; or into thegenomic DNA of a prokaryote or eukaryote, or which exists as a separatemolecule (for example, a cDNA) independent of other sequences. Thenucleotides of the invention can be ribonucleotides,deoxyribonucleotides, or modified forms of either nucleotide. The termincludes single and double forms of DNA.

DNA sequences encoding a VL and/or VH of a humanized antibody thatspecifically binds TAG-72 can be expressed in vitro by DNA transfer intoa suitable host cell. The cell may be prokaryotic or eukaryotic. Theterm also includes any progeny of the subject host cell. It isunderstood that all progeny may not be identical to the parental cellsince there may be mutations that occur during replication. Methods ofstable transfer, meaning that the foreign DNA is continuously maintainedin the host, are known in the art.

Polynucleotide sequences encoding a VL and/or VH of a humanized antibodythat specifically binds TAG-72 can be operatively linked to expressioncontrol sequences. An expression control sequence operatively linked toa coding sequence is ligated such that expression of the coding sequenceis achieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto, appropriate promoters, enhancers, transcription terminators, a startcodon (for instance, ATG) in front of a protein-encoding gene, splicingsignal for introns, maintenance of the correct reading frame of thatgene to permit proper translation of MRNA, and stop codons.

The polynucleotide sequences encoding a VL and/or VH of a humanizedantibody that specifically binds TAG-72 can be inserted into anexpression vector including, but not limited to, a plasmid, virus orother vehicle that can be manipulated to allow insertion orincorporation of sequences and can be expressed in either prokaryotes oreukaryotes. Hosts can include microbial, yeast, insect and mammalianorganisms. Methods of expressing DNA sequences having eukaryotic orviral sequences in prokaryotes are well known in the art. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitation, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with the polynucleotide sequence of interest, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein (see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

Isolation and purification of recombinantly expressed polypeptides maybe carried out by conventional means including preparativechromatography and immunological separations.

Pharmaceutical Compositions and Therapeutic Methods

Pharmaceutical compositions are disclosed herein that include ahumanized CC49 monoclonal antibody, such as V47, V48, V58 or V59, thatcan be formulated with an appropriate solid or liquid carrier, dependingupon the particular mode of administration chosen. In addition, ahumanized CC49 monoclonal antibody linked to an effector molecule (forinstance, toxin, chemotherapeutic drug, or detectable label) can beprepared in pharmaceutical compositions.

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure are conventional. For instance, parenteral formulationsusually comprise injectable fluids that are pharmaceutically andphysiologically acceptable fluid vehicles such as water, physiologicalsaline, other balanced salt solutions, aqueous dextrose, glycerol or thelike. Excipients that can be included are, for instance, other proteins,such as human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered can also contain minoramounts of non-toxic auxiliary substances, such as wetting or emulsifingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate. Actual methods for preparingadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemingtons Pharmaceutical Sciences, 19^(th) Ed., Mack PublishingCompany, Easton, Pa. (1995).

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can include eyedrops, ointments, sprays and the like. Inhalation preparations can beliquid (for example, solutions or suspensions) and include mists, spraysand the like. Oral formulations can be liquid (for example, syrups,solutions or suspensions), or solid (for example, powders, pills,tablets, or capsules). Suppository preparations can also be solid, gel,or in a suspension form. For solid compositions, conventional non-toxicsolid carriers can include pharmaceutical grades of mannitol, lactose,starch, or magnesium stearate. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in the art.

The pharmaceutical compositions that include a humanized CC49 monoclonalantibody, such as V47, V48, V58 or V59, can be formulated in unit dosageform, suitable for individual administration of precise dosages. Inaddition, the pharmaceutical compositions may be administered as animmunoprophylactic in a single dose schedule or as an immunotherapy in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of treatment may be with more than one separate dose, forinstance 1-10 doses, followed by other doses given at subsequent timeintervals as needed to maintain or reinforce the action of thecompositions. Treatment can involve daily or multi-daily doses ofcompound(s) over a period of a few days to months, or even years. Thus,the dosage regime will also, at least in part, be determined based onthe particular needs of the subject to be treated and will be dependentupon the judgment of the administering practitioner. In one specific,non-limiting example, a unit dosage can be about 0.1 to about 10 mg perpatient per day. Dosages from about 0.1 up to about 100 mg per patientper day may be used, particularly if the agent is administered to asecluded site and not into the circulatory or lymph system, such as intoa body cavity, into a lumen of an organ, or directly into a tumor. Theamount of active compound(s) administered will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated.

The compounds of this disclosure can be administered to humans on whosetissues they are effective in various manners such as topically, orally,intravenously, intramuscularly, intraperitoneally, intranasally,intradermally, intrathecally, subcutaneously, via inhalation or viasuppository. The particular mode of administration and the dosageregimen will be selected by the attending clinician, taking into accountthe particulars of the case (for example, the subject, the disease, thedisease state involved, and whether the treatment is prophylactic).

In one embodiment, a therapeutically effective amount of a humanizedCC49 antibody, such as V47, V48, V58 or V59, is the amount of humanizedCC49 antibody necessary to inhibit further growth of a TAG-72-expressingtumor or suppress the growth of a TAG-72-expressing tumor, withouteliciting a HAMA response in the patient receiving the treatment. Inother embodiments, a therapeutically effective amount of humanized CC49antibody is the amount of humanized CC49 antibody necessary to eliminateor reduce the size of a TAG-72-expressing tumor, without eliciting aHAMA response. Specific, non-limiting examples of TAG-72-expressingtumors are adenocarcinoma, colorectal, gastric, pancreatic, breast,lung, and ovarian tumors. In yet another embodiment, a therapeuticallyeffective amount of humanized CC49 antibody is an amount of humanizedCC49 antibody that is effective at reducing a sign or a symptom of thetumor and induces a minimal HAMA response.

A therapeutically effective amount of a humanized CC49 monoclonalantibody, such as V47, V48, V58 or V59, can be administered in a singledose, or in several doses, for example daily, during a course oftreatment. In one embodiment, treatment continues until a therapeuticresult is achieved. However, the effective amount of humanized CC49antibody will be dependent on the subject being treated, the severityand type of the affliction, and the manner of administration of thetherapeutic(s).

Controlled release parenteral formulations of a humanized CC49monoclonal antibody can be made as implants, oily injections, or asparticulate systems. For a broad overview of protein delivery systems(see Banga, A. J., Therapeutic Peptides and Proteins: Formulation,Processing, and Delivery Systems, Technomic Publishing Company, Inc.,Lancaster, Pa., 1995). Particulate systems include microspheres,microparticles, microcapsules, nanocapsules, nanospheres, andnanoparticles. Microcapsules contain the therapeutic protein as acentral core. In microspheres the therapeutic is dispersed throughoutthe particle. Particles, microspheres, and microcapsules smaller thanabout 1 μm are generally referred to as nanoparticles, nanospheres, andnanocapsules, respectively. Capillaries have a diameter of approximately5 μm so that only nanoparticles are administered intravenously.Microparticles are typically around 100 μm in diameter and areadministered subcutaneously or intramuscularly (see Kreuter, J.,Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc.,New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on ControlledDrug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y.,pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec et al., J. Parent. Sci. Tech.44:58, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systemsfor controlled delivery of therapeutic proteins are known (for example,U.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735 and U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206, U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

Site-specific administration of the disclosed compounds can be used, forinstance by applying the humanized CC49 antibody to a pre-cancerousregion, a region of tissue from which a tumor has been removed, or aregion suspected of being prone to tumor development. In someembodiments, sustained intra-tumoral (or near-tumoral) release of thepharmaceutical preparation that includes a therapeutically effectiveamount of humanized CC49 antibody may be beneficial.

The present disclosure also includes therapeutic uses of varianthumanized CC49 monoclonal antibodies, such as V47, V48, V58, or V59,that are non-covalently or covalently linked to effector molecules. Inone specific embodiment, the humanized CC49 monoclonal antibody iscovalently linked to an effector molecule that is toxic to a tumor orcell expressing TAG-72. In one specific, non-limiting example, theeffector molecule is a toxin. In other specific, non-limiting examples,the effector molecule is a radioactive isotope, a chemotherapeutic drug,a bacterially-expressed toxin, a virally-expressed toxin, a venomprotein, or a cytokine.

The cell growth inhibiting chimeric molecules include of a humanizedCC49 monoclonal antibody, such as V47, V48, V58 or V59, linked to atoxin. This molecule can be prepared in pharmaceutical compositions.Toxins can be employed with antibodies that specifically bind TAG-72 toyield immunotoxins. Exemplary toxins include ricin, abrin, diphtheriatoxin and subunits thereof, as well as botulinum toxins A through F.These toxins are readily available from commercial sources (for example,Sigma Chemical Company, St. Louis, Mo.). Diphtheria toxin is isolatedfrom Corynebacterium diphtheriae. Ricin is the lectin RCA60 from Ricinuscommunis (Castor bean). The term also references toxic variants thereofFor example, see, U.S. Pat. No. 5,079,163 and U.S. Pat. No. 4,689,401.Ricinus communis agglutinin (RCA) occurs in two forms designated RCA₆₀and RCA₁₂₀ according to their molecular weights of approximately 65 and120 kD respectively (Nicholson & Blaustein, J. Biochim. Biophys. Acta266:543, 1972). The A chain is responsible for inactivating proteinsynthesis and killing cells. The B chain binds ricin to cell-surfacegalactose residues and facilitates transport of the A chain into thecytosol (Olsnes et al., Nature 249:627-631, 1974 and U.S. Pat. No.3,060,165).

Abrin includes toxic lectins from Abrus precatorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 kD and are composed of two disulfide-linked polypeptide chainsA and B. The A chain inhibits protein synthesis; the B-chain (abrin-b)binds to D-galactose residues (see, Funatsu, et al., Agr. Biol. Chem.52:1095, 1988; and Olsnes, Methods Enzyinol. 50:330-335, 1978).

In several embodiments, the toxin is Pseudomonas exotoxin (PE). The term“Pseudomonas exotoxin” as used herein refers to a full-length native(naturally occurring) PE or a PE that has been modified. Suchmodifications may include, but are not limited to, elimination of domainIa, various amino acid deletions in domains Ib, II and III, single aminoacid substitutions and the addition of one or more sequences at thecarboxyl terminus such as KDEL and REDL. See Siegall et aL, J. Biol.Chem. 264:14256, 1989. In a preferred embodiment, the cytotoxic fragmentof PE retains at least 50%, preferably 75%, more preferably at least90%, and most preferably 95% of the cytotoxicity of native PE. In a mostpreferred embodiment, the cytotoxic fragment is more toxic than nativePE.

Native Pseudomonas exotoxin A (PE) is an extremely active monomericprotein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa,which inhibits protein synthesis in eukaryotic cells. The native PEsequence is provided as SEQ ID NO: 1 of commonly assigned U.S. Pat. No.5,602,095, incorporated herein by reference. The method of action isinactivation of the ADP-ribosylation of elongation factor 2 (EF-2). Theexotoxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2. The function of domain Ib (aminoacids 365-399) remains undefined, although a large part of it, aminoacids 365-380, can be deleted without loss of cytotoxicity. See Siegallet al., J. Biol. Chem. 264:14256-14261, 1989, incorporated by referenceherein.

PE employed includes the native sequence, cytotoxic fragments of thenative sequence, and conservatively modified variants of native PE andits cytotoxic fragments. Cytotoxic fragments of PE include those whichare cytotoxic with or without subsequent proteolytic or other processingin the target cell (for example, as a protein or pre-protein). Cytotoxicfragments of PE include PE40, PE38, PE37, and PE35. PE40 is a truncatedderivative of PE as previously described in the art. See, Pai et al.,Proc. Nat'l Acad. Sci. USA 88:3358-62, 1991; and Kondo et al., J. Biol.Chem. 263:9470-9475, 1988. PE35 is a 35 kD carboxyl-terminal fragment ofPE composed of a methionine at position 280 followed by amino acids281-364 and 381-613 of native PE. PE37, another truncated derivative ofPE, is described in U.S. Pat. No. 5,821,238. PE38 is a truncated PEpro-protein composed of amino acids 253-364 and 381-613 which isactivated to its cytotoxic form upon processing within a cell (see U.S.Pat. No. 5,608,039, incorporated herein by reference). In a particularlypreferred embodiment, PE38 is the toxic moiety of the immunotoxin,however, other cytotoxic fragments, such as PE35, PE37, and PE40, arecontemplated and are disclosed in U.S. Pat. No. 5,602,095; U.S. Pat. No.5,821,238; and U.S. Pat. No. 4,892,827, each of which is incorporatedherein by reference.

Monoclonal antibodies covalently linked to an effector molecule have avariety of uses. For example, a humanized CC49 antibody such as V47,V48, V58 or V59 can be linked to a radioactive isotope and used inimmunotherapy. A humanized CC49 antibody covalently linked to aradioactive isotope is of use to localize a tumor in radioimmunoguidedsurgery, such that the tumor can be removed. In one embodiment, about 10mCi of a radiolabeled humanized CC49 monoclonal antibody is administeredto a subject. In other embodiments, about 15 mCi, about 20 mCi, about 50mCi, about 75 mCi or about 100 mCi of a radiolabeled humanized CC49monoclonal antibody is administered to a subject.

The present disclosure also includes combinations of a humanized CC49monoclonal antibody, such as V47, V48, V58 or V59, with one or moreother agents useful in the treatment of tumors. For example, thecompounds of this disclosure can be administered in combination witheffective doses of immunostimulants, anti-cancer agents,anti-inflammatory agents, anti-infectives, and/or vaccines. The term“administration in combination” or “co-administration” refers to bothconcurrent and sequential administration of the active agents. A subjectthat is suffering from a tumor, or is predisposed to the development ofa tumor, will be a candidate for treatment using the therapeutic methodsdisclosed herein.

Diagnostic Methods and Kits

A method is provided herein for the in vivo or in vitro detection ofTAG-72-expressing tumors or cells. An in vivo detection method canlocalize any tumor or cell that expresses TAG-72 in a subject. In oneembodiment, a diagnostically effective amount of a humanized CC49antibody such as V47, V48, V58 or V59 is administered to the subject fora sufficient amount of time for the antibody to localize to the tumor orcell in the subject and to form an immune complex with TAG-72. Theimmune complex is then detected. In one specific, non-limiting exampledetection of an immune complex is performed by immunoscintography. Otherspecific, non-limiting examples of immune complex detection includeradiolocalization, radioimaging, or fluorescence imaging.

In one example, the antibody is directly linked to an effector moleculethat is a detectable label. Specific, non-limiting examples ofdetectable labels include a radioactive isotope, an enzyme substrate, aco-factor, a ligand, a chemiluminescent agent, a fluorescent agent, ahapten, or an enzyme.

In another example, a diagnostically effective amount of a humanizedCC49 antibody and a secondary antibody are administered to the subjectfor a sufficient amount of time for the humanized CC49 antibody to forman immune complex with TAG-72 on a tumor or cell, and for the secondaryantibody to form an immune complex with the humanized CC49 antibody. Inone embodiment, the humanized CC49 antibody is complexed with thesecondary antibody prior to their administration to the subject. In onespecific, non-limiting embodiment, the secondary antibody is linked to adetectable label. In one embodiment, the immune complex, which includesTAG-72, the humanized CC49 antibody, and the secondary antibody linkedto a detectable label, is detected as described above.

A method of detecting tumors in a subject includes the administration ofa diagnostically effective amount of a humanized CC49 antibody such asV47, V48, V58 or V59 complexed to an effector molecule, such as aradioactive isotope. After a sufficient amount of time has elapsed toallow for the administered radiolabeled antibody to localize to thetumor, the tumor is detected. In one specific, non-limiting example, aradiolabeled immune complex is detected using a hand held gammadetection probe. Primary tumors, metastasized tumors or cells expressingTAG-72 can be detected.

For example, a humanized CC49 antibody complexed to an effectormolecule, such as a radioactive isotope, is administered to a subjectprior to surgery or treatment. In one specific embodiment, the detectionstep is performed prior to surgery to localize the tumor. In anotherembodiment, the detection step is performed during surgery, for exampleto detect the location of the tumor prior to removing it, as inradioimmunoguided surgery. A humanized CC49 antibody complexed to aneffector molecule, such as a radioactive isotope, can also beadministered to a subject following surgery or treatment, to determinethe effectiveness of the treatment, such as to ensure the completeremoval of the tumor, or to detect a recurrence of the tumor.

In vitro detection methods are provided herein. These methods can beused to screen any biological sample to assess for the presence of atumor or cell that expresses TAG-72. A biological sample can be obtainedfrom a mammal, such as a human, suspected of having a tumor expressingTAG-72. In one embodiment the subject has a colorectal tumor. In otherembodiments, the subject has a gastric tumor, a pancreatic tumor, abreast tumor, a lung tumor, an adenocarcinoma, or an ovarian tumor.Other biological samples that can be detected by the in vitro detectionmethod include samples of cultured cells that express TAG-72.

Such samples include, but are not limited to, tissue from biopsies,autopsies, and pathology specimens. Biological samples also includesections of tissues, such as frozen sections taken for histologicalpurposes. Biological samples further include body fluids, such as blood,serum, saliva, or urine.

A kit is provided herein for detecting a TAG-72-expressing tumor orcell. Kits for detecting a TAG-72-expressing tumor or cell willtypically include a humanized CC49 antibody that specifically bindsTAG-72, such as one or more of V47, V48, V58 or V59. An antibodyfragment, such as an Fv fragment can be included in the kit. Theantibody can also be provided as an immunoconjugate. Thus, in severalexamples, the antibody is conjugated to a detectable label, such as aradioactive isotope, an enzyme substrate, a co-factor, a ligand, afluorescent agent, a hapten, an enzyme, or a chemiluminescent agent.

The kit can further include instructional materials disclosing means ofuse of an antibody that specifically binds TAG-72, such as V47, V48, V58or V59, or a fragment thereof. The instructional materials may bewritten, in an electronic form (for example, computer diskette orcompact disk) or may be visual (for example, video files). The kits canalso include additional components to facilitate the particularapplication for which the kit is designed. Thus, for example, the kitmay additionally contain a means of detecting a label (for example,enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). In one example, the kit contains a secondaryantibody that is conjugated to a detectable label. Kits can additionallyinclude buffers and other reagents, such as an antigen (for example,purified TAG-72) routinely used for the practice of a particular method,or of use in the preparation of a suitable control. Such kits andappropriate contents are well known to those of skill in the art.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Methods Used to Generate Variant HuCC49V10 Antibodies

In the experiments disclosed herein, the dispensability of some of themurine framework residues that were deemed crucial and consequentlyretained in HuCC49V10 (V10) has been tested. Several new variants of V10were generated by replacing, using site-specific mutagenesis, some ofthe murine framework residues with their counterparts in the humantemplates. The variants were tested for their (a) Ag-binding reactivityand (b) reactivity to Abs in sera from patients who had earlier beenadministered mCC49. One such variant, V59, contains only three murineresidues in its VL and VH frameworks versus 19 murine residues presentin the parental Ab V10. Compared with V10, V59 shows a significantlylower reactivity to the anti-V region antibodies present in thepatients' sera, while its Ag-binding affinity is unexpectedly comparableto that of the parental antibody. mAb V59 exhibits low sera reactivityand is therefore a more useful clinical reagent against human carcinomasthan its predecessors. Thus, as disclosed herein, humanization of an Abcan be experimentally optimized, in terms of maintaining Ag binding andminimizing immunogenicity, by the judicious manipulation of frameworkresidues.

The following methods were used in the experimental studies:

Synthetic oligonucleotides: Oligonucleotide primers listed below wereused for the site-specific mutagenesis of the VL and VH domains of theAb V10. V10 is described in U.S. patent application Ser. No. 09/830,748,filed Apr. 30, 2001, (herein incorporated by reference). V10 has beendeposited with ATCC on Aug. 28, 2003 and has ATCC Accession No.PTA-5416. They were supplied by Gene Probe Technologies (Gaithersburg,Md.), but could be obtained from a variety of commercial sources. Themutagenic bases are underlined, the positions of the residue changes areparenthetically enclosed, and the sequences recognized by restrictionendonucleases are in bold italics. VL primers: 3′ VL (5):5′-GTCTGGAGACTGGGTCATCACGATG-3′ (SEQ ID NO: 53) 3′ VL (19, 21):5′-GGACTTGCAATTGATAGTGGCCCTCTCGCG-3′ (SEQ ID NO: 54) 5′ VH (43):5′-CCAGGGCAGCCTCCTAAACTGCTG-3′ (SEQ ID NO: 55) 3′ VL (43):5′-CAGCAGTTTAGGAGGCTGCCCTGG-3′ (SEQ ID NO: 56) 5′ VH (78):5′-CAATCAGCAGCCTGCAGGCAGAAG-3′ (SEQ ID NO: 57) 3′ VL (106): 5′-GCAG

CCCGTTTGATTTCCAGCTTGGTGCC-3′ (SEQ ID NO: 58) 3′ VL (100):5′-CAGCTTGGTGCCCTGGCCGAAGCTGAGG-3′ (SEQ ID NO: 59) 3′ VL (100, 106):5′-GCAGCCCCGTTTGATTTCCAGCTTGGTGCCCTGGCC-3′ (SEQ ID NO: 60) VH primers:3′ VH (12): 5′-CCCCAGGTTTCTTCACCTCAGCGC-3′ (SEQ ID NO: 61) 3′ VH (20):5′-CCTTGCAGGACACCTTCACGGAAGC-3′ (SEQ ID NO: 62) 5′ VH (38, 48):5′-CCACTGGGTGAGACAGAATCCTGGACAGCGCCTGGAGT (SEQ ID NO: 63)GGATGGGATATTTCTC-3′ 3′ VH (38, 48):5′-GAGAAATATCCCATCCACTCCAGGCGCTGTCCAGGATT (SEQ ID NO: 64)CTGTCTCACCCAGTGG-3′ 5′ VH (40): 5′-GGTGAGACAGGCTCCTGGACAGC-3′ (SEQ IDNO: 65) 5′ VH (66, 67, 69): 5′-GTTCCAGGGCAGGGTGACCATCACTGCAGACAC-3′ (SEQID NO: 66) 3′ VH (66, 67, 69): 5′-GTGTCTGCAGTGATGGTCACCCTGCCCTGGAAC-3′(SEQ ID NO: 67) 5′ VH (80): 5′-GCACTGCCTACATGGAGCTCTCCAGC-3′ (SEQ ID NO:68) 5′ VH (93): 5′-GTGTACTTCTGCGCCAGATCCCTGAATATG-3′. (SEQ ID NO: 69)

The sequences of the 20- to 25-bp-long end primers that were used forDNA amplification of the desired VL and VH genes were as follows: (SEQID NO: 70) 5′ VL: 5′-GC

CCACCATGGATA-3′ (SEQ ID NO: 71) 5′ VH: 5′-CTA

CACCATGGAGTGGTCC-3′ (SEQ ID NO: 72) 3′ VH: 5′-ATGGGCCCGTAGTTTTGGCGC-3′

Each of the 5′ primers carries a restriction endonuclease site (HindIIIfor VL and EcoRI for VH), followed by the Kozak consensus sequence and asequence encoding the N-terminus of the signal peptide. The 3′ VL endprimer (either primer SEQ ID NO: 6 or 8) contains a unique Sacil sitelocated 10 bp downstream from the start of the human κ C region, whilethe 3′ VH primer carries a unique ApaI site located 17 bp downstreamfrom the start of the human CH1 domain.

DNA mutagenesis and sequencing: All PCR amplifications were carried outin EasyStart™ 50 tubes (Molecular Bio-Products, San Diego, Calif.) in afinal volume of 50 μl of PCR buffer (20 mM Tris-HCl (pH 8.4), 50 mM KCl)containing 2 mM MgCl₂, 200 μM of dNTPs, 2.5 units of the high-fidelitycopy PfuTurbo DNA polymerase (Stratagene Cloning Systems, La Jolla,Calif.), 0.1% Triton X-100, 25 pmoles each of the appropriate 5′ and 3′primers, and 100 ng of the DNA template.

Initial denaturation at 95° C. for 7 minutes was followed by 30 cyclesof denaturation at 95° C. for 30 seconds, annealing at 55° C. for 30seconds and extension at 72° C. for 1 minute, and a final primerextension step at 72° C. for 10 minutes.

The VL and VH genes of the framework variants of V10 were generatedusing a two-step PCR method as described by Landt et al. (Gene96:125-128, 1990).

The first PCR was carried out using a mutagenic oligomer as one of thetwo primers to incorporate a desired change, while the mutated productof the first PCR was used as one of the primers for the second PCR.After an initial denaturation at 95° C. for 7 minutes, 30 cycles ofdenaturation (95° C., 1 minute), primer annealing (45° C., 1 minute) andextension (72° C., 2 minutes) were followed by a final extension for 10minutes at 72° C. When the mutations were located in the middle of theVL or VH gene, an overlap extension PCR was used to incorporate thealterations (Higuchi et al., Nucleic Acids Res 16:7351-7367, 1988; Ho etal., Gene 77:51-59, 1989). Two separate PCRs were performed to amplifytwo halves of the complete gene. The resulting PCR fragments wereannealed and amplified using the appropriate 5′ and 3′ end primers.

Expression vector and generation of expression constructs: Prior tocloning into expression vectors, the synthesized VL or VH genes weresequenced using the ABI PRISM™ dRhodamine terminator cycle sequencingkit (Perkin Elmer Applied Biosystems, Foster City, Calif.). Themammalian expression vector pDCMdhfr (FIG. 1) was used for theco-expression of the Ig L and H chains in Chinese hamster ovary (CHO)cells. The pDCMdhfr vector was constructed by joining the NruI and PvuIdouble-digested DNA fragments of the vectors, kc-dhfrΔE-S (Ryu et al.,Hum Antibodies Hybridomas 7:113-122, 1996) and pDCM, a construct derivedfrom pcDNA3 (Invitrogen, Carlsbad, Calif.). pDCMdhfr contains cloningsites for the two target genes, each downstream from anenhancer-promoter complex of the immediate early genes of human CMV. Theplasmid also includes a dhfr expression unit driven by anenhancer-deficient SV40 early promoter, and a neomycin resistance genefor drug selection of transfectants.

To generate a pDCMdhfr expression construct of the genes encoding the Land H chains of the V10 parental Ab, a construct containing the L chainwas first generated (FIG. 1A). This was done by sequential digestion ofthe pre-existing construct pBScHuCC49V5 (Tamura et al., J Immunol164:1432-1441, 2000) with EcoRI, mung bean nuclease, and HindIII. V5encodes the light chain gene of the parental mAb V10. It was followed bythe ligation of the resulting V5 DNA fragment into the pDCMdhfr vectorthat has been pre-treated with ApaI, mung bean nuclease, and HindIII. ADNA fragment encoding the H chain gene of the mAb V10 was generated byClaI digestion, addition of the NotI linker, and EcoRI/NotI digestion ofthe pre-existing construct pBScHuCC49V8 (Tamura et al., J Immunol164:1432-1441, 2000). The resulting V8 DNA fragment, which encodes theheavy chain of V10, was cloned into the EcoRI/NotI site of the pDCMdhfrvector containing the V10 L chain (FIG. 1B). To generate expressionconstructs encoding the framework variants of the V10 L chains, themutated VL region sequences were exchanged with the VL sequence of V10in the pDCMdhfr L chain construct through the HindIII/SacII site, priorto insertion of the H chain gene into the vector. In the pDCMdhfrconstruct containing both L and H chains, SacII is not a unique sitebecause it is present in the DNA sequence in the C region of the Hchain. To generate constructs encoding the H chains of the variants, thePCR-amplified VH sequences were swapped with the V10 VH sequence in thepDCMdhfr construct containing both L and H chains, using the uniqueEcoRI/ApaI site.

Mammalian cell culture and production of recombinant Abs: To developtransfectants expressing V10 and its framework variants, CHOdhfr⁻ cellswere transfected with the pDCMdhfr derived expression construct usingliposome-mediated DNA transfer (Lipofectamine Plus, Invitrogen)according to the guidelines of the manufacturer. Following transfection,cells were incubated at 37° C. in DMEM/F12 medium overnight, and werethen trypsinized and seeded in 96-well plates at 2×10⁴ cells per well inselection medium (alpha MEM, 10% dialyzed fetal bovine serum, 550 μg/mlG418). After 2 weeks of selection, the culture supernatants of thestable transfectants were monitored by ELISA assay and Western blotting.

Purification of recombinant Abs: The highest Ab-producing clones weregrown in CHO-S-SFM II serum-free medium (Invitrogen) supplemented with550 μg/ml G418. Cell culture supernatants were collected and centrifugedat 2,000×g for 10 minutes to remove cellular debris. The supernatant wasthen loaded on a protein A agarose column (Invitrogen) equilibrated in20 mM Tris-HCl buffer (pH 7.5). The bound protein was eluted from thecolumn with 0.1 M glycine hydrochloride (pH 2.5) and the pH of theeluted material was immediately adjusted to 7.4 with 1.0 M Tris (pH8.0). The protein was concentrated using Centricon 30 (Amicon, Beverly,Mass.) and buffer-exchanged in PBS (pH 7.4). The protein concentrationwas determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories,Hercules, Calif.) based on the method developed by Bradford (Bradford,Anal Biochem 72:248-254, 1976). The purity of the Ab preparation wasevaluated using the Agilent 2100 Bioanalyzer system (AgilentTechnologies, Waldronn, Germany), under reducing and non-reducingconditions, using the Protein 200 LabChip kit (Agilent Technologies).

ELISA: ELISA assays were carried out by coating 96-well polyvinylmicrotiter plates with 1 μg/well of bovine submaxillary mucin (3SM)(Sigma-Aldrich, St. Louis, Mo.). The plates were blocked with 5% BSA inPBS for 1 hour at 37° C. and then washed with 1% BSA in PBS. Fiftymicroliters of culture supernatants were loaded in duplicate wells.After a 1 hour incubation at 37° C. and washing with 1% BSA in PBS, 100μl of peroxidase-conjugated anti-human IgG (Fcγ-fragment specific)diluted (1:3000) in 1% BSA in PBS was added per well. The plates wereincubated for another hour at 37° C. They were washed prior to detectionusing 100 μl SureBlue™ TMB 1-component peroxidase substrate (KPL,Gaithersburg, Md.). The calorimetric reaction was allowed to proceed for10 minutes at room temperature in the dark, before it was terminated bythe addition of 100 μl of TMB Stop solution (KPL) per well. Theabsorbance was read at 450 nm.

Empirical comparative analysis of Ag binding via SPR: The Ag reactivityof the Abs was measured by SPR, using a BIAcore X instrument (BIAcore,Piscataway, N.J.). BSM was immobilized on the carboxymethylated dextranCM5 sensor chips by amine coupling (Johnsson et al., Anal Biochem198:268-277, 1991; Schuck et al., Current Protocols in Protein Science2:20.2.1-21, 1999). The dextran layer of the sensor chip was activatedby injecting 35 μl of a mixture of N-ethyl-N′-(3dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimideat a flow rate of 5 μl/minute. BSM, diluted in 100 mM sodium acetatebuffer (pH 3.0) at a concentration of 100 μg/ml, was injected until asurface of 1000 resonance units (RUs) was obtained. The remainingreactive groups on the surface were blocked by injecting 35 μl of 1 Methanolamine (pH 8.5). Binding measurements were performed at 25° C. inHBS running buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA and0.005% Tween 20). Abs (100 μl), in increasing concentrations, wereapplied sequentially at a flow rate of 5 μl/minute, and the associationat each concentration was monitored for 20 minutes. The BSM surface wasregenerated, using a 1 minute injection of 6 M guanidine and 0.2 Macetic acid without any loss of the BSM-surface activity.

Competition RIA: The relative Ag-binding affinity of V10 and itsvariants was determined using a competition RIA as described previously(Tamura et al., J Immunol 164:1432-1441, 2000). Twenty-five μl of serialdilutions of the Abs to be tested as well as mCC49 (positive control)and HuIgG (negative control), prepared in 1% BSA in PBS, were added tomicrotiter plates containing 10 ng of BSM saturated with 5% BSA in PBS.¹²⁵I-labeled mCC49 (100,000 cpm in 25 μl) was then added to each well.After an overnight incubation at 4° C., the plates were washed and thebound radioactivity was counted in a γ-scintillation counter. Therelative affinity constants were calculated by a modification of theScatchard method (Frankel et al., Mol Immunol 16:101-106, 1979).

Flow cytometric (FACS) analysis: To evaluate the ability of V10 and itsvariants to bind to cell-surface TAG-72, a previously described method(Nicolet et al., Tumour Biol 18:356-366, 1997) was used for FACSanalysis. Jurkat cells (1×10⁶), expressing TAG-72, were resuspended incold Ca⁺⁺ and Mg⁺⁺ free Dulbecco's PBS and incubated with the Abs (V10or variants) for 30 minutes on ice. A human IgG was used as an isotypecontrol. After one washing cycle, the cell suspension was stained withFITC-conjugated mouse anti-human Ab (PharMingen, San Diego, Calif.) for30 minutes on ice. After a second washing cycle was performed, thesamples were analyzed with a FACScan (Becton Diclinson, Mountain View,Calif.) using CellQuest for Macintosh. Data from the analysis of 10,000cells were obtained.

Immunoadsorption of patient serum: Stored patients' sera EA and DS, froma Phase I clinical trial (Mulligan et al., Clin Cancer Res 1:1447-1454,1995) that involved the administration of ¹⁷⁷Lu-labeled mCC49 topatients with advanced adenocarcinoma, were used to assess the serareactivity of the V10 derived Abs. Sera from patients EA and DS thatwere found to contain anti-V region Abs to CC49 (Iwahashi et al., MolImmunol 36:1079-1091, 1999) also contained circulating TAG-72 antigenand anti-murine Fc Abs, which could interfere with the binding of theV10 and its variants to the sera anti-V region Abs. To overcome thisdifficulty, TAG-72 and Abs to murine Fc were removed from the sera byimmunoadsorption prior to checking the sera reactivity of V10 and itsvariants. The procedure for immunoadsorption has been detailed earlier(Iwahashi et al., Mol Immunol 36:1079-1091, 1999). Briefly, purifiedmurine CC92, a second-generation mAb that reacts with an epitope ofTAG-72 distinct from the one recognized by mCC49 (Koroki et al., CancerRes 50:4872-4879, 1990), was coupled to Reacti-gel (HW65F; Pierce,Rockford, Ill.) (Hearn et al., J Chromatogr 185:463-470, 1979). Serumwas added to an equivalent volume of the CC92 gel (wet-packed volume)and incubated overnight at 4° C. with end-over-end rotation. The sampleswere centrifuged at 1000×g for 5 minutes and the supernatant was savedand stored at −20° C.

Sera reactivity: The reactivity of V10 and its variants to anti-V regionAbs was determined using a recently developed SPR-based competitionassay (Gonzales et al., J Immuizol Methods 268:197-210, 2002).Competition experiments were performed at 25° C. using a CM5 sensor chipcontaining HuCC49 in flow cell 1 and rabbit gamma globulin (Bio-Rad), asa reference, in flow cell 2. HuCC49, V10 or its variants were used atdifferent concentrations to compete with the HuCC49 immobilized on thesensor chip for binding to serum anti-V region Abs. Patient's serum withor without the competitor (HuCC49, V10 or its variants) was appliedacross the sensor surface using a recently developed sample applicationtechnique (Abrantes et al., Anal Chem 73:2828-2835,2001). The sample wascentered across the sensor surfaces and an oscillatory flow was appliedat a rate of 20 μl/minute. After measuring the binding for 1000 seconds,the unbound samples were removed from the surfaces by washing withrunning buffer using a flow rate of 100 μl/min, and the surfaces wereregenerated with a 1 minute injection of 10 mM glycine (pH 2.0). Thepercent binding at each Ab concentration was calculated as follows:% binding=[slope of the signal obtained with competitor(serum+Abs)/slope of the signal obtained without competitor (serumonly)]×100.The IC₅₀ for each antibody, the concentration required for 50%inhibition of the binding of the serum anti-V region Abs to immobilizedHuCC49, was calculated.

Example 2 Design and Generation of the Genes Encoding the V10 FrameworkVariants

An examination of the VL sequences of mCC49 and the human Ab LEN revealsthat 62 out of the 80 framework residues are identical (Kashmiri et al.,Hybridoma 14:461-473, 1995). Of the 18 differences, 7 residues weredeemed crucial and were grafted onto the human template, along with theCDRs or the SDRs, in generating the humanized Abs HuCC49 and V10,respectively. The VH sequences of mCC49 and 21/28′CL share 63 identitiesin 87 framework residues. Out of the 24 differences, 12 were retained asmurine in the humanization protocols of CC49. The murine frameworkresidues retained in the V domains of the HuCC49 and V10 Abs are atpositions 5, 19, 21, 43, 78, 100 and 106 in VL, and at positions 12, 20,38, 40, 48, 66, 67, 69, 71, 80, 91 and 93 in VH (numbering convention ofKabat et al. (Kabat et al., Sequence of Proteins of ImmunologicalInterests, 5^(th) ed., p. NIH Publication No. 91-3242, U.S. Departmentof Health and Human Services, National Institutes of Health, Bethesda,Md., 1991)). Analysis of the known three-dimensional structures of Ab:Agcomplexes available in the PDB database (Abola et al., Methods Enzymol277:556-571, 1997) reveals that the residues at the positions enumeratedabove are probably important in keeping the overall structure of thecombining site, because they are either buried or implicated in thedirect interaction with the Ag (Amit et al., Science 233:747-753, 1986;Colman et al., Nature 326:358-363, 1987; Fischmann et al., J Biol Chem266:12915-12920, 1991; Padlan et al., Proc Natl Acad Sci USA86:5938-5942, 1989; Sheriff et al., Proc Natl Acad Sci USA 84:8075-8079,1987; Tulip et al., J Mol Biol 227:122-148, 1992), contact with CDRs(Chothia et al., J Mol Biol 196:901-917, 1987; Chothia et al., Nature342:877-883, 1989; Tramontano et al., J Mol Biol 215:175-182, 1990), orin the VL/VH interaction (Padlan, Mol Immunol 31:169-217, 1994). Thecrucial nature of these murine framework residues, however, has not beenvalidated in the case of CC49. It is probable that some of the frameworkresidues deemed crucial to maintain Ag reactivity of the other Abs arenot that essential for the Ag:Ab interaction of CC49. Several variantsof mAb V10 were designed to test the indispensability of some of themurine framework residues for the Ag-binding reactivity of CC49 byreplacing them with the corresponding residues in the human Abs.

Genes encoding the VL and VH domains of the variants were generated byprimer-induced mutagenesis, using the pBScHuCC49V5 and pBScHuCC49V8constructs (Tamura et al., J Immunol 164:1432-1441, 2000), which containthe L and H chains of V10, respectively, as the templates. The V regionsequences were synthesized either by a dual step PCR or an overlapextension PCR, as described in Materials and Methods. Each of the Vregions of the L and H chains was generated by the primer-inducedmutation, inserted into the pDCMdhfr expression cassette, as describedin Materials and Methods, and subsequently sequenced. V35 was generatedby replacing the murine VL framework residues 5, 19, 21, 78 and 106 ofV10 with the corresponding residues of the human Ab LEN. V37 containstwo additional changes at positions 43 and 100, making all the frameworkresidues in the VL of this variant human. To generate V40, the VHframework residues 20, 38, 48, 66, 67, 69 and 80 of the V10 werereplaced with the corresponding residues of the human Ab 21/28′CL. Inaddition to all the mutations present in V40, mutations at positions 12and 40 were also included in V41. In this VH variant, only three murineresidues located at positions 71, 91 and 93 of the murine Ab have beenretained. The amino acid sequences of the VL and VH frameworks of theparental V10 and the mutated variants are shown in FIG. 2. Variantscontaining different combinations of the parental and variant L and Hchains are listed in Table II. TABLE II Residue positions substituted inthe HuCC49V10 framework variants Variant Light Chain Heavy Chain V10None None (parent) V35 5, 19, 21, 78, 106 None V37 5, 19, 21, 43, 78,100, 106 None V40 None 20, 38, 48, 66, 67, 69, 80 V41 None 12, 20, 38,40, 48, 66, 67, 69, 80 V47 5, 19, 21, 78, 106 20, 38, 48, 66, 67, 69, 80V48 5, 19, 21, 43, 78, 100, 106 20, 38, 48, 66, 67, 69, 80 V58 5, 19,21, 78, 106 12, 20, 38, 40, 48, 66, 67, 69, 80 V59 5, 19, 21, 43, 78,100, 106 12, 20, 38, 40, 48, 66, 67, 69, 80

Example 3 Expression of V10 Variants in CHOdhfr⁻ Cells

The expression constructs of the genes encoding the H and L chains ofthe parental mAb V10 and its framework variants were introduced intoCHOdhfr⁻ cells. The supernatants harvested from the G418 resistanttransfectants were assayed for Ig production by ELISA and Western blotanalysis as described in Materials and Methods. Most of thetransfectants, like those generated by the control constructs of V10,were found to be positive for Ig production. When the culturesupernatants were assayed for their reactivity to TAG-72, most of theV10 variants, like those of V10 transfectomas, were positive. Thehighest producing clone of each construct was cultured under identicalconditions, and the secreted Abs were purified from the culturesupernatants. The purity of the Ab preparations was verified by theAgilent 2100 Bioanalyzer system, using a Protein 200 LabChip. Theprofiles of all recombinant Abs were identical under reducing andnon-reducing conditions. Under reducing conditions (FIG. 3), all Absyielded two protein bands of approximately 24-27 kDa and 60 kDa. Thesemolecular masses are in conformity with those of the Ig L and H chains,respectively.

Example 4 Binding of V10 Framework Variants to TAG-72-Positive BSM

To assess the binding reactivity of the framework variants to theTAG-72⁺ BSM, an SPR-based assay was carried out using a sensor chip withBSM immobilized on its surface. Increasing concentrations of differentAbs were allowed to interact with the BSM surface for 20 minutes.Responses could be measured after the binding of the antibody to thesurface BSM reaches equilibrium, but a steady state could not beattained for most of the samples, especially at the higherconcentrations. Nonetheless, the magnitude of the response signals couldprovide a measure of the binding interaction of different Abs to the BSMsurface.

Results from the binding measurements revealed that different frameworkvariants generate distinctly different profiles of binding to thesurface BSM. An examination of the binding profiles suggests that allthe framework variants have retained their reactivity to BSM (FIG. 4).Higher response signals by some of the variants than that generated byV10 may suggest that some variants have higher affinity for the Ag thanthat of the parental Ab. When a sensor chip immobilized with anti-humanFcγ specific Ab was used, the binding profiles of all the frameworkvariants to the anti-human Fcγ sensor chip were not different from eachother. Thus, the differences in the signals generated by differentvariants against BSM are due to their binding reactivity to BSM, and notbecause of differences in their protein concentrations.

The complexity of the surface-binding reaction of whole antibodies,attributed mainly to their bivalency, precludes a detailedinterpretation of the time-course of binding, as is frequently appliedto SPR biosensor data (Schuck, Biophys Biomol Struct 26:541-566, 1997).In addition, the multiplicity of Ab binding sites on the BSM surface[disaccharide and trisaccharide structural epitopes (Hanisch et al.,Biol Chem Hoppe Seyler 370:21-26, 1989)) complicated the analysis of thebiosensor data. Therefore, it is not possible to calculate the exactrelative affinity constants of the variants from their binding profilesgenerated by the SPR-based assay. Nevertheless, a simple comparison ofthe binding signals at defined concentrations for each antibody gave asemi-quantitative measure of its surface-binding reactivity.

To determine the relative affinity constants of the variants,competition RIA was carried out. Only two of the variants, V59 and V47,were compared to the parental Ab V10. These two variants were selected,because the latter generated the highest response profile in theSPR-based assay, while the former carries the largest number of humanresidues in its frameworks. Competition RIA, which included mCC49 andHuCC49 as positive controls and an irrelevant human IgG as a negativecontrol, was performed as described in Example 1. Serial dilutions ofunlabeled Abs were used to compete with the binding of ¹²⁵I-mCC49 toBSM. Results presented in FIG. 5 show that the competition profile ofthe V47 variant is shifted slightly to the right, while that of V59slightly to the left of the V10 profile. Compared to 110 ng of V10 Ab,Briefly, 217 ng of V47 and 85 ng of V59 (compared to 110 ng of V10) wererequired for 50% inhibition of the binding of ¹²⁵I-mCC49 to BSM (TableIII). TABLE III Relative binding affinities of framework variants viacompetition RIA. Amount of Ab Required Ka (relative affinity AntibodyName for 50% Inhibition (ng) constant) × 10⁸ M⁻¹ mCC49 33 1.98 HuCC49 551.16 V10 110 0.59 V47 217 0.32 V59 85 1.08

These results are in conformity with the values of the relative affinityconstants (K_(a)) of V10, V47 and V59 that were found to be 0.59×10⁸M⁻¹, 0.32×10⁸ M⁻¹ and 1.08×10⁸ M⁻¹, respectively (Table III). Theaffinity constants were calculated from the linear parts of thecompetition curves shown in FIG. 5. The relative affinity constants ofmCC49 and HuCC49 were found to be 1.98×10⁸ M⁻¹ and 1.16×10⁸M⁻,respectively.

The competition RIA results seem to be at variance with the data in FIG.4 in which V47 shows the highest binding response to BSM using theSPR-based assay. It should be noted, however, that the competition RIAwas carried out at low concentrations of Abs (highest concentration usedwas 135 nM) to reflect the binding of the Abs to the high-affinitybinding site in BSM. The higher binding signals of V47 by SPR wereevident only at high Ab concentrations (500 and 1250 nM), which reflectthe binding of V47 to the low-affinity binding sites in BSM.

Example 5 Binding of V10 Variants to Cell Surface TAG-72

Flow cytometric analysis was used to measure the binding of V10, V47 andV59 to the TAG-72 expressed on the cell surface of Jurkat cells (Nicoletet al., Tumour Biol 18:356-366, 1997). When 0.5 μg of each Ab was used,no significant differences were found in the mean fluorescenceintensity, or in the percentage of Jurkat cells that were reactive withV10 and its framework variants (FIG. 6). V59 and V47 showed 29.0% and27.6% of gated cells and the mean fluorescence intensities of 49.8 and56.8, respectively, compared with V10, which showed 25.8% of gated cellsand a mean fluorescence intensity of 62.9. Thus, the binding of the twoframework variants to cells displaying TAG-72 on their cell surfaces wasnot significantly different from that of the parental Ab V10.

Example 6 Reactivity of V10 and its Framework Variants to Patients' Sera

To assess the potential immunogenicity of V47 and V59, relative to V10,the Abs were evaluated for their in vitro reactivity to sera fromadenocarcinoma patients who were treated with ¹⁷⁷Lu-mCC49 in a phase Iclinical trial (Mulligan et al., Clin Cancer Res 1:1447-1454, 1995). Asdescribed in Example 1, any circulating TAG-72 and anti-murine Fc Abswere removed from the sera by immunoadsorption with mCC92, a murine Abof the same isotype as that of mCC49, that reacts with an epitope ofTAG-72 different from the one recognized by mCC49. Previous studies haveshown that the pre-adsorbed sera from patients EA and DS contain anti-Vregion Abs to mCC49 (Gonzales et al., J Immunol Methods 268:197-210,2002; Iwahashi et al., Mol Immunol 36:1079-1091, 1999; Tamura et al., JImmunol 164:1432-1441,2000). Sera reactivity of HuCC49, V10 and itsvariants was determined by their ability to compete with HuCC49immobilized on a sensor chip for binding to the mCC49 anti-V region Abspresent in the patient's serum. The IC₅₀ value, the concentration of thecompetitor Ab required for 50% inhibition of the binding of immobilizedHuCC49 to the patient's serum, was calculated by plotting the percentbinding as a function of competitor concentration. A higher IC₅₀ valueindicates a decreased reactivity to the serum, suggesting reducedpotential immunogenicity of the Abs in patients. FIG. 7 shows thecompetition profiles generated by HuCC49, V10 and its variants when theywere used to compete with the HuCC49 immobilized on the sensor chip forbinding to the anti-V region Abs present in the sera of patients EA(FIG. 7A) and DS (FIG. 7B). The pattern of the competition profiles forthe two patients' sera was similar. The variant V47 shows the samereactivity as V10, whereas V59 demonstrates a weaker reactivity to thesera, as reflected by the shift in the competition curves to the right.The IC₅₀ value of V59 for the serum EA is about 10-fold and 35-foldhigher than that of V10 and HuCC49, respectively (Table IV). TABLE IVConcentrations of competitor antibody required for half- maximalinhibition (IC₅₀) of binding of patients' sera to HuCC49 immobilized onthe sensor chip Competitor Antibody Patient EA (nM) Patient DS (nM)HuCC49 2.2 6.0 V10 7.7 >1000 V47 8.2 >1000 V59 82.5 >1000

For the serum DS, the binding of HuCC49 to the serum anti-V region Abswas only minimally inhibited by V10 and V59. The IC₅₀ values could notbe determined because even 1000 nM of the variants failed to achievemore than 30% inhibition of the binding of sera antibodies to the HuCC49immobilized on the sensor chip. Still, the competition profile of V59 isshifted slightly to the right of V10. Compared to 80 nM of V10 or V47,approximately200 nM of V59 is required for 20% inhibition of the bindingof the sera anti-V region Abs to HuCC49 immobilized on the sensor chip(FIG. 7B, Inset). This corresponds to a 2.5-fold decrease in thereactivity of V59 to the DS serum, compared to V10. Compared to HuCC49,which caused 20% binding inhibition at 2 nM concentration, theinhibition by V59 is 100-fold less.

The two most important and often contrary aims in humanizing an Ab are(a) retention of its Ag-binding property and (b) reducing itsimmunogenicity in patients. For the retention of ligand-bindingproperty, the functional conformation of the Ab-combining site must bepreserved, which requires maintenance of the CDRs and their interactionwith each other and with the rest of the structure of theantibody-combining site. Successful humanization of an Ab, therefore,depends on selecting the most appropriate human templates based onsequence identity and optimization of the acceptor frameworks to retainthose murine framework residues that may be crucial in maintaining thestructural integrity of the combining site. Unfortunately, optimizationof the frameworks increases the risk of making the Ab immunogenic inpatients. The more murine framework residues are retained in a humanizedAb, the more immunogenic it is likely to be. Framework residues areconsidered crucial when they (i) are involved in direct interaction withthe Ag (Amit et al., Science 233:747-753, 1986; Colman et al., Nature326:358-363, 1987; Fischmann et al., J Biol Chem 266:12915-12920, 1991;Padlan et al., Proc Natl Acad Sci USA 86:5938-5942, 1989; Sheriff etal., Proc Natl Acad Sci USA 84:8075-8079, 1987; Tulip et al., J Mol Biol227:122-148, 1992), (ii) contact the CDRs affecting the structure oftheir loops and, consequently, the Ag-binding site (Chothia et al., JMol Biol 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989;Tramontano et al., J Mol Biol 215:175-182, 1990), (iii) are involved inVL/VH interaction (Padlan, Mol Immunol 31:169-217, 1994), and (iv) areburied and influence the overall structure of the combining site. In theabsence of a three-dimensional structure of the Ab:Ag complex, as in thecase of mCC49, selecting the crucial framework residues to be retainedin the humanized Ab is based on the analysis of the known crystalstructures of other Ab:Ag complexes that are available in the PDBdatabase (Abola et al., Methods Enzymol 277:556-571, 1997). As shownherein, data obtained by studying the effect of site-specific mutationson the ligand-binding property of a particular Ab provides definiteinformation as to which framework residues are absolutely essential forits Ag-binding property (compared to the information obtained from thePDB database). This approach helps to exclude all those murine frameworkresidues from the humanized Ab that are dispensable for the Ag-bindingactivity and led to the development of antibodies that elicited only aminimal immune response in patients.

The humanized Ab, V10, was generated by grafting only the SDRs of CC49onto the VL and VH frameworks of human Abs LEN and 21/28′CL,respectively, while retaining those murine framework residues that werepresumed essential, according to the PDB database, for its Ag-bindingactivity. The dispensability of some of the murine framework residuesthat were retained in V10 was tested in the experiments disclosed hereinby replacing them with their counterparts in human templates andevaluating the Ag-binding activity of the resulting variants.

Framework variants of V10 were designed to study the effect of severalmutations (substitutions of human for the murine residues) concurrently,instead of generating mutants containing individual changes. Abscontaining different combinations of VL and VH variants were alsogenerated. ELISA and SPR assays show that all of the framework variantsretain reactivity to TAG-72 positive BSM. Competition RIA further showsthat the Ag-binding affinity of the combination variant V59 wascomparable to that of V10. The variant V59, the most humanized variantof V10, contains only three murine residues in its VH frameworks,compared with 19 murine residues in the VL and VH frameworks of V10.This translates to an increase in the human residue content to 91.2% inV59 from 84.2% in V10. These results lead to the conclusion that the VLframeworks of CC49 can be replaced fully with those of the human Ab LENwithout any loss of activity; only positions 94 and 96 in LCDR3 havebeen retained as murine in the L chain of V59. Furthermore, the humansubstitutions in the VH at positions 12, 20, 38, 40, 48, 66, 67, 69 and80 do not adversely affect the Ag-binding affinity of the Ab.

Whether the V59 variant displays reduced potential to evoke anti-Vregion response in patients as opposed to the CDR-grafted HuCC49 or V10was assessed by comparing their reactivity to the sera fromadenocarcinoma cancer patients who were administered ¹⁷⁷Lu-CC49 in aPhase I clinical trial (Mulligan et al., Clin Cancer Res 1:1447-1454,1995). The sera were shown to carry anti-V region antibodies to CC49(Gonzales et al., J Immunol Methods 268:197-210, 2002; Iwahashi et al.,Mol Immunol 36:1079-1091, 1999; Tamura et al., J Immunol 164:1432-1441,2000). The results show that V59, compared with V10, was less reactiveto two patients' sera, EA and DS.

Thus, the humanization of the V10 Ab has been augmented by replacingmurine framework residues, initially deemed crucial in maintaining thecombining site structure, with the corresponding human residues.Judicious manipulation of framework residues, disclosed herein, has ledto the optimization of the humanization of an Ab and has been used toenhance its biological properties. The experiments disclosed herein showthat, for the first time, manipulation of framework residues candecrease the sera immune reactivity of Abs.

Example 7 Clinical Studies

A non-blinded, single-arm pilot “phase I” evaluation of ¹²⁵Iradiolabeled are V47, V48, V58, and/or V59 monoclonal antibody (mAb) inthe intraoperative detection of disease in human subjects undergoingsurgery for metastatic colorectal cancer is performed. During thesurgical procedure, detection or localization of the antibody in primaryor recurrent tumor is performed using the Neoprobe® hand-heldgamma-detecting probe. The primary objective is to determine the timeinterval from antibody injection to surgery that minimizes the timeinterval from injection to surgery while insuring that no more than ⅓(one-third) of patients fail to demonstrate tumor localization.

Prior to surgery, patients are injected with 2 mCi of ¹²⁵I radiolabeledto 1 mg of mAb by intravenous injection. Beginning two days prior toinjection of the antibody, patients are given a saturated solution ofpotassium iodide (SSKI) 10 drops daily to block thyroid uptake of theradioiodinated mAb. Thyroid blocking is continued for two weekspostinjection.

To determine the optimum time interval from injection to surgery amodified ‘standard method’ 3 by 3 phase I design is used. A cohort ofthree patients initially undergoes surgery at 3 days (72 hours)following injection. If all three patients localize the metastatictumor, the time interval is reduced to two days (48 hours) betweenantibody injection and surgery. If one of three patients fails tolocalize, an additional three patients are treated at that timeinterval. If two or more of six patients fail to localize, it isdetermined if failure to localize is due to either high backgroundcounts obscuring tumor-bound antibody or low overall counts reflectingrapid clearance of both circulating and bound antibody. If the failureis due to high counts, the next cohort of three patients is treated atan interval from injection to surgery increased to four days (96 hours).If failure is due to low overall counts, the next cohort has theirinterval reduced to two days (48 hours). This three patient cohort isevaluated as the previous cohort. If all three patients localize, theinterval is reduced to one day (24 hours) from injection to surgery.Patients do not have their interval from injection to surgery reduced toless than one day (24 hours) or greater than seven days (168 hours).

The surgeon initially explores the abdomen using traditional techniques(visually and manually) to determine the presence or absence of tumor.Following exploration, the surgeon uses the gamma-detecting probe deviceto obtain triplicate, 2-second counts of non-lymphatic tumor(s)identified by traditional techniques. A biopsy of at least one tumoridentified by traditional surgical techniques is obtained if possible.The gamma-detecting probe device is used to obtain counts of the tumormargin. All counts are taken in triplicate.

Abdominal exploration is carried out to assess areas of abnormalradioactive uptake using a gamma detecting probe. Attempts are made toprocure as much tumor tissue and tumor associated antigen lymphatictissue as possible for sampling. Biopsies of the liver, lymph nodes andperitoneum (particularly the momentum) are obtained when appropriate. Afinal status of residual radioactivity is recorded at the completion ofthe surgical procedure.

Pharmacokinetic parameters are determined by noninvasive dailyprecordial counts obtained using a Neoprobe® 1000 probe for a period ofseven days following injection of the radiolabeled monoclonal antibody.Quantitative 24-hour urine specimens are collected for 72 hoursfollowing injection. This initial study requires not fewer than 12patients or more than 30 patients.

Data is analyzed to determine the optimal time from injection to surgerybased on tumor localization. Safety data on adverse events associatedwith the mAb administration and surgical procedure are reported. Theoptimum time interval is then recommended for further study.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. A humanized anti-TAG-72 CC49 monoclonal antibody comprising: a lightchain comprising a light chain Complementarity Determining Region(L-CDR)1, a L-CDR2, a L-CDR3, and a light chain framework region fromHuCC49V10, a heavy chain comprising a heavy chain ComplementarityDetermining Region (H-CDR)1, a H-CDR2, a H-CDR3, and a heavy chainframework region from HuCC49V10, wherein the light chain frameworkregion comprises a corresponding framework residue from human antibodyLEN at position 5, 19, 21, and 106 in the light chain, and wherein theheavy chain framework region comprises a corresponding framework residuefrom human antibody 21/28′CL at positions 20, 38, 48, 66, 67, 69, and 80in the heavy chain; wherein the humanized CC49 antibody retains bindingaffinity for TAG-72 and has reduced immunogenicity, as compared to aparental humanized CC49 V10 antibody.
 2. The monoclonal antibody ofclaim 1, further comprising a corresponding human LEN framework residueat position 43 in the light chain.
 3. The monoclonal antibody of claim1, further comprising a corresponding human LEN framework residue atposition 78 in the light chain.
 4. The monoclonal antibody of claim 1,further comprising a corresponding LEN human framework residue atposition 100 in the light chain.
 5. The monoclonal antibody of claim 1,further comprising a corresponding human 21/28′CL framework residue atposition 12 in the heavy chain.
 6. The monoclonal antibody of claim 1,further comprising a corresponding human 21/28′CL framework residue atposition 40 in the heavy chain.
 7. The monoclonal antibody of claim 1,wherein the light chain framework region further comprises acorresponding human LEN framework residue at position 43, 78, and 100 inthe light chain and a corresponding human 21/28′CL framework residue atposition 12 in the heavy chain.
 8. The monoclonal antibody of claim 1,wherein L-CDR1 comprises an amino acid sequence set forth as SEQ ID NO:9, L-CDR2 comprises an amino acid sequence set forth as SEQ ID NO: 10,and L-CDR3 comprises an amino acid sequence set forth as SEQ ID NO: 11.9. The monoclonal antibody of claim 1, wherein H-CDR1 comprises an aminoacid sequence et forth s SEQ ID NO: 12, H-CDR2 comprises an amino acidsequence set forth as SEQ ID NO: 13, and H-CDR3 comprises an amino acidsequence set forth as SEQ ID NO:
 14. 10. The monoclonal antibody ofclaim 1, wherein the light chain framework comprises SEQ ID NO: 1 andwherein the residue at position 5 in the light chain is a threonine, theresidue at position 19 in the light chain is an alanine, and the residueat position 21 in the light chain is an isoleucine.
 11. The monoclonalantibody of claim 1, wherein the light chain framework comprises anamino acid sequence set forth as SEQ ID NO: 2 and wherein the residue atposition 43 in the light chain is a proline.
 12. The monoclonal antibodyof claim 1, wherein the light chain framework comprises an amino acidsequence set forth as SEQ ID NO: 3 and wherein the residue at position78 in the light chain is a leucine.
 13. The monoclonal antibody of claim1, wherein the light chain framework comprises an amino acid sequenceset forth as SEQ ID NO: 4 and wherein the residue at position 100 in thelight chain is a glutamine and the residue at position 106 in the lightchain is an isoleucine.
 14. The monoclonal antibody of claim 1, whereinthe heavy chain framework comprises an amino acid sequence set forth asSEQ ID NO: 5 and wherein residue at position 20 in the heavy chain is avaline.
 15. The monoclonal antibody of claim 14, wherein the heavy chainframework comprises an amino acid sequence set forth as SEQ ID NO: 5 andwherein residue at position 12 in the heavy chain is a lysine.
 16. Themonoclonal antibody of claim 1, wherein the heavy chain frameworkcomprises an amino acid sequence set forth as SEQ ID NO: 6 and whereinthe residue at position 38 in the heavy chain is an arginine and theresidue at position 48 in the heavy chain is a methionine.
 17. Themonoclonal antibody of claim 16, wherein the heavy chain frameworkcomprises an amino acid sequence set forth as SEQ ID NO: 6 and whereinthe residue at position 40 in the heavy chain is an alanine.
 18. Themonoclonal antibody of claim 1, wherein the heavy chain frameworkcomprises an amino acid sequence set forth as SEQ ID NO: 7 and whereinthe residue at position 66 in the heavy chain is an arginine, theresidue at position 67 in the heavy chain is an isoleucine, and theresidue at position 80 in the heavy chain is a methionine.
 19. Themonoclonal antibody of claim 1, wherein the heavy chain frameworkcomprises the amino acid sequence set forth as SEQ ID NO:
 8. 20. Themonoclonal antibody of claim 1, further comprising a detectable label.21. The monoclonal antibody of claim 1, further comprising an effectormolecule.
 22. The monoclonal antibody of claim 20, wherein the label isa fluorescent or a radioactive molecule.
 23. The monoclonal antibody ofclaim 21, wherein the effector molecule is a toxin.
 24. A compositioncomprising a functional fragment of the humanized monoclonal antibody ofclaim 1, wherein the functional fragment specifically binds TAG-72. 25.The composition of claim 24, wherein the fragment comprises an Fv, anFab, or an F(ab′)₂.
 26. The composition of claim 25, wherein theantibody is encoded by a nucleic acid sequence as deposited as ATCCPTA-5415.
 27. A pharmaceutical composition comprising a therapeuticallyeffective amount of the antibody of claim 1 in a pharmaceuticallyacceptable carrier.
 28. A method for treating a subject with a tumorthat expresses TAG-72, comprising: administering a therapeuticallyeffective amount of the humanized antibody of claim 1 to the subject,thereby treating the tumor.
 29. The method of claim 28, wherein thehumanized antibody is encoded by a nucleic acid sequence as deposited asATCC PTA-5415.
 30. A method for detecting a cell expressing TAG-72 in asubject, comprising contacting a sample from the subject with theantibody of claim 1, and detecting the presence of a complex of theantibody with TAG-72, thereby detecting a cell expressing TAG-72. 31.The method of claim 30, wherein the subject has a tumor.
 32. The methodof claim 31, wherein the antibody is labeled.
 33. The method of claim30, wherein the antibody is encoded by a nucleic acid deposited as ATCCPTA-5415.
 34. The method of claim 30, wherein the sample is a biopsyspecimen, autopsy specimen, and pathology specimens, or a biologicalfluid.
 35. A method for in vivo diagnosis of cancer in a subject,comprising (a) administering to an mammal a diagnostically effectiveamount of the antibody of claim 20, (b) allowing sufficient time for theantibody to become specifically localized to at least one cancer cell,and (c) detecting the labeled antibody in vivo at a site where theantibody has become localized, thereby diagnosing the cancer.
 36. A kitcomprising a container comprising the humanized antibody of claim 1 andinstructions.
 37. A monoclonal antibody, comprising a heavy and a lightchain variable region, wherein the light chain variable region comprisesa light chain framework region comprising amino acid sequences set forthas SEQ ID NOs: 41-44, and light chain complementarity determiningregions comprising amino acid sequences set forth as SEQ NOs: 9-12; theheavy chain variable region comprises a heavy chain framework regioncomprising amino acid sequences set forth as SEQ ID NOs: 49-52, andheavy chain complementarity determining regions comprising amino acidsequences set forth as SEQ ID NOs: 12-14; and wherein the humanized CC49antibody retains binding affinity for TAG-72 and has reducedimmunogenicity, as compared to a parental humanized CC49 V10 antibody.38. An isolated nucleic acid encoding the antibody of claim
 1. 39. Avector comprising a promoter operably linked to the nucleic acid ofclaim
 38. 40. A host cell transformed with the vector of claim
 39. 41.The host cell of claim 40, wherein the cell is a eukaryotic cell. 42.The antibody of claim 1, further comprising a tyrosine to prolinesubstitution in L-CDR3 at position
 91. 43. The antibody of claim 42,further comprising a valine to leucine substitution at position 27b.